# Patent

## Title
Peptide having ability to activate cancer-related gene

## Abstract

To provide a cancer diagnostic reagent for determining malignancy of a cancer patient or a cancer cell and a tendency of canceration of a healthy subject, the reagent including a peptide having an ability to activate a cancer-related gene and extracted from cell membrane surfaces of human squamous-cell carcinoma cells or including a synthetic polynucleotide encoding the peptide or a partial amino acid sequence of the peptide.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to peptides activating a cancer-related gene and derived from cell membrane surfaces of human cancer cells, immunosuppressive agents including the peptides as an effective component, and anticancer agents including antibodies against the peptides as an effective component. The present invention further relates to diagnostic reagents including polynucleotides encoding the peptides for diagnosing malignancy of cancer or tendency of canceration. Furthermore, the present invention relates to methods for manufacturing the peptides.
2. Description of the Related Art
Cancer is a disease caused by cells which started unregulated growth. The cells continue to proliferate and infiltrate into neighboring normal cells to destroy the normal functions. Some of the cells spread by metastasis from their original site to one or more sites elsewhere in the body, leading to a loss of normal cell functions and to depression of functions of organs. Thus, cancer is a disease that leads patients to death. Normal cells proliferate but will stop growing when they come in contact with solid substances (contact inhibition). On the other hand, the contact inhibition property is lost in cancerous cells. Consequently, the cancerous cells continue to grow in their host as long as the host is alive. Among cells extracted from the body, cells having ability for continuing unlimited growth can be established as a cell line. Cancer, in a broad sense, is a state of uncontrolled cell growth due to mutation of a gene, in particular, due to mutation of a gene positively or negatively regulating cell growth. Heretofore, many studies have been conducted on genes contributing to carcinogenesis or a growth mechanism and peptides or proteins relating to them (Japanese Unexamined Patent Application Publication Nos. 2003-517306 and 2000-217585).
Among peptides or proteins derived from cell membrane surfaces of human cancer cells, generally, HLA-binding peptides are particularly thought to be cancer antigens and are thought to act on immunocompetent cells as antigens, most of all, as immunogens. Additionally, it is widely known that some proteins and glycoproteins derived from cancer cells and immunocompetent cells such as macrophages isolated from a cancer-bearing living body have an immunosuppressing activity preventing the destruction of cancer cells, for example, immunosuppressive acidic protein (IAP). Such proteins and glycoproteins are clinically used for measuring the degree of immunosuppression.
Heretofore, it has been thought that cancer cell growth progresses by a mechanism due only to self division of cancerous cells. However, the rate of cancer cell growth is not constant and cancer cells rapidly proliferate at some point. Such phenomena cannot be fully explained by the conventional view only.
SUMMARY OF THE INVENTION
On the basis of the above-mentioned phenomenon that cancer rapidly progresses at some point, cancer cells have a possibility of secreting a factor outside the cancer cells. Such a factor further accelerates the cancer cell growth by acting on normal cells neighboring the cancer cells so as to activate a cancer-related gene in the normal cells. If this factor is found, not only a mechanism of progression of cancer is clarified but also a useful drug can be provided.
Namely, it is an object of the present invention to provide a factor having a function of accelerating cancer cell growth by acting on normal cells neighboring the cancer cells so that a cancer-related gene in the normal cells is activated and to provide a corresponding gene of the factor. It is another object of the present invention to develop a valuable use of the factor and its corresponding gene by verifying the factor for the cancer-related-gene-activating function in normal cells.
The present inventor has performed intensive studies for overcoming the above-mentioned problems, and, as a result, found a peptide and a gene encoding the peptide in an extract of a human squamous-cell carcinoma cell line UTC-8 which has a high activity of metastasis. Furthermore, the inventor has proved that the peptide has a function of activating a cancer-related gene in normal cells neighboring cancer cells by acting on the normal cells and accelerating cancer growth, and has proved that the peptide can be used as an immunosuppressive agent. In addition, the inventor has found that a polynucleotide capable of hybridizing with the gene encoding the peptide can be used as a reagent for diagnosing malignancy of cancer or measuring tendency of canceration (easiness of conversion to cancer: a degree of risk of canceration), which was not included in clinical examination items before, and that an antibody against the peptide has an anticancer activity. Thus, the present invention has been accomplished.
The present invention relates to aspects (1) to (10) described below:
(1) A peptide having an ability to activate a cancer-related gene, wherein the peptide is derived from cell membrane surfaces of human squamous-cell carcinoma cells and includes an amino acid sequence represented by SEQ ID No: 1 or an amino acid sequence having deletion, substitution, or addition of one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1;
(2) A peptide having an ability to activate a cancer-related gene, wherein the peptide is derived from cell line UTC-8 (FERM BP-08611) established from human squamous-cell carcinoma; and the peptide shows a detectable peak at a detection wavelength of 214 nm in gel filtration of an extract from the cell line;
(3) An immunosuppressive agent comprising the peptide according to the aspect (1) or (2) as an effective component;
(4) An antibody against the peptide according to the aspect (1) or (2);
(5) A diagnostic reagent including the antibody according to the aspect (4) for determining tendency of canceration or malignancy of cancer;
(6) An anticancer agent comprising the antibody according to the aspect (4) as an effective component;
(7) A polynucleotide including a nucleotide sequence encoding at least three contiguous amino acids of the amino acid sequence of the peptide according to the aspect (1);
(8) A diagnostic kit for determining tendency of canceration or malignancy of cancer, wherein the kit includes a peptide according to the aspect (1) or (2) and a polynucleotide according to the aspect (7);
(9) The diagnostic kit according to the aspect (8) further including at least one marker gene selected from the group consisting of:
Ras oncogene family;
v-crk avian sarcoma virus CT10 oncogene homolog-like lactate dehydrogenase B;
Placental growth factor;
Interleukin 8;
MAS1, activator of S phase kinase;
v-raf;
v-fms;
v-rel;
v-src;
GRO1;
Hepatoma-derived growth factor;
Vascular endothelial growth factor;
Bone morphogenic protein 3;
Squamous-cell carcinoma antigen recognized by T cell;
Interleukin-1 beta;
Conserved gene amplified in osteosarcoma; and
Lymphoid blast crisis oncogene; and
(10) A method for preparing a peptide according to the aspect (1) or (2), the method including the steps of immersing cells obtained by culturing human squamous-cell carcinoma cells in a culture medium into a citrate-phosphate buffer having a pH of 3.3 to 3.4 for extraction; and fractionating the extract by gel filtration using a citrate-phosphate buffer having a pH of 6.8 to 7.0 for yielding a peptide having an ability to activate a cancer-related gene in human normal cells.
The peptide according to the present invention is derived from cell membrane surfaces of human squamous-cell carcinoma cells. In particular, the peptide is extremely remarkable in the fact that it has ability to activate a cancer-related gene in human normal cells. Since the sensitivity of cells and tissues against this peptide of the present invention can be an indicator to predict canceration tendency of normal cells or progress of cancer, the peptide can be a useful diagnostic reagent for determining tendency of canceration or malignancy of cancer. Additionally, it is strongly suggested that the unregulated growth of cancer cells is caused by, not only cancer cells themselves, but also the interaction accompanying the activation of various cancer-related genes in normal cells. Thus, an entirely novel mechanism of cancer growth is presented.
Therefore, the present invention can contribute greatly to the research and development of diagnosis and therapy of cancer.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B are elution patterns of gel filtration of peptides extracted in Example 1 according to the present invention. FIG. 1A shows elution patterns of the peptides by using a citrate-phosphate buffer having a pH of 7.2 as an elution buffer and detection wavelengths of 280 nm and 210 nm. FIG. 1B shows elution patterns of the fraction (elution volume: 21 ml) of the rightmost peak of the elution pattern using the detection wavelength of 280 nm in FIG. 1A by using a citrate-phosphate buffer having a pH of 6.8 as an elution buffer and detection wavelengths of 280 nm and 214 nm.
FIGS. 2A and 2B are electrophoresis photographs showing gene pattern changes in cells treated with a peptide of the present invention which were investigated by conducting a PCR reaction using cDNA prepared by reverse transcription of total mRNA derived from the cells treated with the peptide as a template and using DNA encoding the full length of the peptide of the present invention as a primer.
FIGS. 3A and 3B are electrophoresis photographs showing gene pattern changes in cells treated with a peptide of the present invention. The gene was prepared by a PCR reaction using cDNA prepared by reverse transcription of total mRNA derived from the cells treated with the peptide as a template and using DNA encoding a sequence consisting of the first to seventh amino acids of the peptide of the present invention as a primer.
FIG. 4 is a photograph showing the result when the monoclonal antibody against the peptide of the present invention was subjected to a cytotoxic test using cancer cells.
FIG. 5 is a diagram showing an immunosuppressive activity of the peptide of the present invention in a transplantation test.



DESCRIPTION OF THE PREFERRED EMBODIMENTS
The peptide according to the present invention has a function of activating a human cancer-related gene and includes the following amino acid sequence:

Gln-Pro-Gln-Phe-Gly-Arg-Arg-Met-Glu-Ser-Lys  (SEQ ID NO: 1)

Additionally, a peptide including an amino acid sequence having deletion, substitution, or addition of one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1 is included in the present invention as long as the peptide has the function of activating a cancer-related gene.
The above-mentioned cancer-related gene refers to not only oncogenes but also immune system genes. The activation of a cancer-related gene means acceleration of canceration or cancer growth through activation of an oncogene or through inactivation of a tumor suppressor gene and/or activation or inactivation of an immune system gene, e.g., a cellular immunity system and/or humoral immunity system.
The immunosuppressive activity of the peptide according to the present invention is notable. For example, it is recognized that the peptide suppresses the rejection in tissue transplantation. Therefore, the peptide of the present invention can be used as an immunosuppressive agent.
The peptide represented by SEQ ID NO: 1 is derived from cell membrane surfaces of human squamous-cell carcinoma cells and can be extracted from human squamous-cell carcinoma cell line UTC-8 (Deposition No.: FERM BP-08611, which is deposited with International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-1 Higashi, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan, on Feb. 4, 2004) by using a citrate-phosphate buffer having a pH of 3.3 to 3.4.
More specifically, after the extraction by the citrate-phosphate buffer having a pH of 3.3 to 3.4, the extract is further applied to gel filtration using a citrate-phosphate buffer having a pH of 6.8 to 7.2 as an elution buffer at a column flow rate of 0.1 to 0.28 ml/min, and then a fraction having a peak in a detection wavelength of 214 nm is isolated to yield the peptide represented by SEQ ID NO: 1.
The peptide represented by SEQ ID NO: 1 of the present invention is characterized by the fact that the peak can be separated and detected only when a detection wavelength of 214 nm is used. The peak of the peptide of the present invention cannot be detected when a detection wavelength of 254 to 257 nm or of 280 nm, which are generally used in peptide detection, is used.
In addition to the above-mentioned extraction method, the peptide according to the present invention can be prepared by conventional chemical synthesis of peptides on the basis of the amino acid sequence. Namely, an amino acid derivative having a carboxyl group and a side-chain functional group being protected by a protecting group and an amino acid derivative having an amino group and a side-chain functional group being protected by a protecting group are condensed in the presence of carbodiimide or the like. Then, the protecting group for the amino group is removed to bind a next amino acid derivative protected by a protecting group. Such a reaction can be performed by a liquid-phase method or a solid-phase method. In general, the solid-phase method is used, except when a relatively large amount of peptide is synthesized. Particularly, in a method developed for identifying an antigenic determinant defined by its amino acid sequence, a spacer arm having an amino group on its tip is used. The spacer arm is prepared by chemically treating a cellulose film or the tip of a plastic pin. In the latter method, a peptide chain is extended by sequential reactions of solutions in a 96-well plate; this method is called multi-pin peptide synthesis.
Furthermore, in another method, a peptide of the present invention may be prepared by chemically synthesizing a DNA encoding the peptide by using a DNA synthesizer; preparing a recombinant vector by connecting the DNA to an appropriate expression vector; introducing the vector into a host such as Escherichia coli; culturing the host; and collecting the peptide of the present invention from the culture.
The peptide of the present invention is useful by itself and can be used as a diagnostic reagent for determining tendency of canceration (easiness of conversion to cancer: a degree of risk of canceration) of normal cells or tissues, for determining whether or not a subject is suffering from cancer, and for determining malignancy of cancer.
The above-mentioned various determinations are performed by bringing the peptide of the present invention in contact with a human cell or tissue specimen obtained by surgical resection or biopsy, and utilizing this contact for detecting a cancer-related gene or for determining a change in the expression degree or the expression pattern of the gene. The sensitivity of cells or tissues against the peptide of the present invention reflects, for example, a tendency of canceration (easiness of conversion to cancer: a degree of risk of canceration) of the cells or tissues or a degree of malignancy of cancer.
In the determination of the degree of malignancy of cancer cells or tissues, the amount of expression of the gene encoding the peptide of the present invention may be directly determined without the contact of the peptide of the present invention with the cells or tissues. However, it is desirable that the cancer cells or tissues are brought into contact with the peptide of the present invention, in order to achieve higher sensitivity in measurement.
Additionally, as is clear in diagnosis methods 1 and 2 described below, a polynucleotide (DNA and RNA) encoding at least three contiguous amino acids of the amino acid sequence of the peptide of the present invention can be used as a diagnostic reagent for determining tendency of canceration (easiness of conversion to cancer: a degree of risk of canceration) of normal cells or tissues, for determining whether or not a subject is suffering from cancer, and for determining malignancy of cancer. A diagnostic kit of the present invention preferably utilizes such a polynucleotide in combination with a peptide of the present invention.
Diagnosis methods using the peptide of the present invention will now be described.
[Diagnosis Method 1]
First, a specimen such as cells and tissues obtained from a subject to be tested is brought into contact with the peptide of the present invention, and they are cultured for a predetermined period of time. Examples of the specimen include cells and tissues obtained from a high risk group for cancer, such as subjects living in area polluted with a chemical or radioactive material and smokers; cells and tissues which are suspected to be a precancerous stage, such as polyp; cells and tissues neighboring cancer cells; cells and tissues at a region under a danger of metastasis of cancer; and peripheral blood.
When the specimen has a tendency of canceration, even if the cells and tissues are normal now, a change in an expression pattern of the gene is observed: the gene encoding the peptide of the present invention is detected, or the amount of expression of the gene is increased. When the malignancy of cancer is high, the amount of expression of the gene is further increased. Therefore, the tendency of canceration or malignancy of cancer can be determined by detecting the gene or determining the degree of its expression.
In this diagnosis method, the cells or tissues of interest are brought into contact with the peptide, and then total mRNA is extracted from the cells or tissues. After synthesis of cDNA by reverse transcription using the mRNA as a template, PCR is further conducted by using the resulting cDNA as a template. In the latter PCR, all polynucleotides encoding sequences consisting of at least three contiguous amino acid residues of the amino acid sequence represented by SEQ ID NO: 1 are collectively used as primers. The polynucleotide may encode the full-length polypeptide.
This diagnosis method will now be specifically described.
First, total mRNA is extracted by a common method from a specimen which is prepared by treating normal tissues or cells with the peptide. Then, cDNA is synthesized by reverse transcription using the total mRNA as templates to generate a cDNA pool which will be used as templates.
DNA encoding a sequence consisting of, for example, the first to fourth amino acids in the amino acid sequence represented by SEQ ID NO: 1 has the following nucleotide sequence:

CAR CCN CAR TTY  (SEQ ID NO:2)

(wherein N is A, T, C, or G, R is G or A, and Y is C or T). Each of the nucleotide sequences, i.e., 32 types of DNA, is synthesized, and a pool including each DNA in an equal amount is used as forward primers.

Since the cDNA used as the template has a poly-T sequence corresponding to poly-A tailing of mRNA, an oligo-dT primer (poly-A primer) is used as a reverse primer.
The PCR using the cDNA pool as the templates is conducted by using the above-mentioned forward primers and the reverse primer, followed by electrophoresis and fluorescence staining.
When a change in an expression pattern of genes including the gene encoding the peptide of the present invention is induced by treating a specimen with the peptide of the present invention, it is indicated that the specimen is sensitive to the treatment with the peptide of the present invention; thus, it is determined that the specimen has a high tendency of canceration even if the cells or tissues as the specimen are currently normal. When a specimen is already cancerous, a change in the gene expression pattern increases with a degree of the malignancy. Therefore, the degree of the malignancy can be determined by measuring the change.
The amount of the peptide expression can be determined by measuring the fluorescence intensity of the band in the electrophoresis. In order to precisely determine the amount of the peptide expression, for example, quantitative PCR may be performed.
The quantitative PCR can be performed by, for example, real-time PCR using the cDNA pool as templates, the above-mentioned primers, and a fluorescent dye or a fluorescence-labeled probe which binds with double-strand DNA. Then, a relationship between the number of the PCR cycles and the fluorescence intensity is determined. By comparing the results with those obtained by using a standard, the amount of the cDNA, i.e., the expression amount of the peptide of the present invention, can be quantitatively determined.
A change in the gene expression pattern or an increase in the peptide expression amount can be determined by comparing the expression level of a control. As the control, the results obtained by conducting the same procedure as above using the same cells except that the peptide of the present invention is not used and/or the results obtained by treating normal cells or tissues that are completely free from cancer risk with the peptide and conducting the same procedure as above, are used.
The normal cells and tissues that are free from cancer risk are derived from young subjects. As such cells, for example, a kidney mesangium cell (ACBRI-1376; Applied Cell Biology Research Institute), a skin fibroblast cell (Catlog2F0-C25; Cell Systems), and a pancreatic epithelial cell (CBRI515; Applied Cell Biology Research Institute) are commercially available.
From the views described above, it is obvious that a combination of the peptide of the present invention and the group of the above-mentioned primers, i.e., the group of polynucleotides encoding sequences consisting of at least three contiguous amino acid residues of the amino acid sequence represented by SEQ ID NO: 1, is useful as a cancer diagnostic reagent kit for determining a tendency of canceration or malignancy of cancer.
[Diagnosis method 2]
In another diagnosis method using the peptide of the present invention, cancer-related gene is used as a marker gene.
In this diagnosis method, cells or tissues of interest are treated with the peptide of the present invention as in the above-mentioned diagnosis method 1; thus the same specimen as in the diagnosis method 1 can be used. However, in this diagnosis method, the reverse transcription is conducted to generate a cDNA (first-strand cDNA) pool, which is complementary to the mRNA, by using total mRNA extracted from the cells or tissues treated with the peptide as a template and using oligo-dT primer having T7 RNA polymerase promoter region as a reverse primer. Then, after the second-strand cDNA synthesis, in vitro transcription (IVT) was conducted using T7 RNA polymerase and biotinylated rNTPs at 37° C. for 14 hr to yield biotin-labeled cRNA. The amount of the cRNA is measured with a spectrophotometer to confirm that at least 10 μL of cRNA can be yielded.
The diagnosis method further includes processes for bringing this cRNA labeled with fluorescence dye or the like into contact with cDNA of a cancer-related gene which is immobilized on a plate for hybridization and for measuring the fluorescence intensity.
When the cRNA is hybridized to some cancer-related gene, it is suggested that the cancer-related gene is being expressed in the cells or tissues. When the fluorescence intensity of some cancer-related gene is increased or decreased, it is suggested that the expression amount of the cancer-related gene is increased or decreased by the treatment with the peptide of the present invention. Namely, for example, when the expression of an oncogene is increased or the expression of an immune system gene which suppresses canceration is decreased, it is suggested that the tendency of canceration is high. Thus, on the basis of the expression condition of a cancer-related gene, a tendency of canceration or malignancy of cancer can be determined.
An increase or decrease in the expression amount of a cancer-related gene is determined by comparing the expression level in a control. The cells and tissues used as the control are the same as those used in the diagnosis method 1.
As described above, immune system genes relating to cancer, in addition to oncogenes, are included in the cancer-related genes in this specification. Examples of such genes are as follows:
Ras oncogene family;
v-crk avian sarcoma virus CT10 oncogene homolog-like lactate dehydrogenase B;
Placental growth factor;
Interleukin 8;
MAS1, activator of S phase kinase;
v-raf;
v-fms;
v-rel;
v-src;
GRO1;
Hepatoma-derived growth factor;
Vascular endothelial growth factor;
Bone morphogenic protein 3;
Squamous-cell carcinoma antigen recognized by T cell;
Interleukin-1 beta;
Conserved gene amplified in osteosarcoma; and
Lymphoid blast crisis oncogene.
At least one gene of these cancer-related genes is used in this invention. For higher precision, yet more cancer-related genes may be used. In such a case, it is preferable to use a combination of genes in the following groups as a genes set for diagnosis.
(a) Oncogene expression-increasing group: a gene encoding the peptide of the present invention and Ras oncogene and c-fos oncogene, etc.;
(b) Immune gene expression-increasing group: Interleukin-1 beta, etc.; and
(c) Immune gene expression-decreasing group: MHC class II, DM, and alpha, beta protein gene and killer cell lectin-like receptor subfamily B, M member 1 gene.
Tables 1 and 2 show examples of cancer-related genes of which expression is increased and immune system genes of which expression is increased or decreased by the treatment with the peptide (HPLC-purified peptide) derived from cell membrane surfaces of human squamous-cell carcinoma cells UTC-8 (Deposition No.: FERM BP-08611, which is deposited with International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology).







TABLE 1





Cancer-related gene enhanced by the HPLC-purified peptide



















Gene Name
Fold Change
Common
Description
Product





K03218
3.912448639
SRC; ASV; SRC1;
v-src sarcoma (Schmidt-Ruppin A-2)
proto-oncogene tyrosine-protein




c-SRC; p60-Src
viral oncogene homolog (avian)
kinase SRC


NM_001201
3.73617959
BMP3
bone morphogenetic protein 3
bone morphogenetic protein 3





(osteogenic)
(osteogenic) precursor


D16431
3.595973492
HDGF
hepatoma-derived growth factor
hepatoma-derived growth factor





(high-mobility group protein 1-like)
(high-mobility group protein 1-like)


X54936
3.576227427
PGF
placental growth factor, vascular
placental growth factor, vascular





endothelial growth factor-related
endothelial growth factor-related





protein
protein


M95712
3.033826351
BRAF
v-raf murine sarcoma viral oncogene
v-raf murine sarcoma viral oncogene





homolog B1
homolog B1


NM_005850
3.023255825
SF3B4
splicing factor 3b, subunit 4, 49
splicing factor 3b, subunit 4





kDa


NM_005937
2.995664358
MLLT6
myeloid/lymphoid or mixed-lineage
myeloid/lymphoid or mixed-lineage





leukemia (trithorax homolog,
leukemia (trithorax homolog,





Drosophila); translocated to, 6
Drosophila); translocated to, 6


NM_001419
2.950166225
ELAVL1
ELAV (embryonic lethal, abnormal
ELAV-like 1





vision, Drosophila)-like 1 (Hu





antigen R)


NM_002613
2.86612194
PDPK1
3-phosphoinositide dependent
3-phosphoinositide dependent





protein kinase-1
protein kinase-1


NM_014308
2.849243879
P101-PI3K
phosphoinositide-3-kinase,
phosphoinositide-3-kinase,





regulatory subunit, polypeptide
regulatory subunit, polypeptide





p101
p101


D26120
2.833239079

splicing factor 1


X61498
2.832505941
NFKB2
nuclear factor of kappa light
nuclear factor of kappa light





polypeptide gene enhancer in
polypeptide gene enhancer in





B-cells 2 (p49/p100)
B-cells 2 (p49/p100)


X03663
2.780905962
CSF1R
colony stimulating factor 1
colony stimulating factor 1





receptor, formerly McDonough
receptor precursor





feline sarcoma viral (v-fms)





oncogene homolog


NM_004204
2.756244421
PIGQ
phosphatidylinositol glycan,
phosphatidylinositol glycan, class





class Q
Q isoform 2;






phosphatidylinositol glycan, class






Q isoform 1


NM_003017
2.741738006
SFRS3
splicing factor, arginine/serine-
splicing factor, arginine/serine-





rich 3
rich 3


NM_000753
2.7408638
PDE3B
phosphodiesterase 3B, cGMP-
phosphodiesterase 3B, cGMP-





inhibited
inhibited


NM_002712
2.696629047
PPP1R7
protein phosphatase 1, regulatory
protein phosphatase 1, regulatory





subunit 7
subunit 7


NM_004906
2.543901205
WTAP
Wilms tumor 1 associated protein
Wilms' tumour 1-associating protein






isoform 1; Wilms' tumour






1-associating protein isoform 2


NM_005207
2.497231245
CRKL
v-crk sarcoma virus CT10 oncogene
v-crk sarcoma virus CT10 oncogene





homolog (avian)-like
homolog (avian)-like


NM_007279
2.493814707
U2AF2
U2 small nuclear ribonucleoprotein
U2 small nuclear ribonucleoprotein





auxiliary factor (65 kD)
auxiliary factor (65 kD)


NM_015714
2.480620146
G0S2
putative lymphocyte G0/G1 switch
putative lymphocyte G0/G1 switch





gene
gene


NM_001511
2.476593256
CXCL1
chemokine (C-X-C motif) ligand 1
chemokine (C-X-C motif) ligand 1





(melanoma growth stimulating





activity, alpha)


NM_016263
2.465854406
FZR1
Fzrl protein
Fzrl protein


NM_012103
2.459948301
AUP1
ancient ubiquitous protein 1
ancient ubiquitous protein 1






isoform 1; ancient ubiquitous






protein 1 isoform 2; ancient






ubiquitous protein 1 isoform 3


M32977
2.456767944
VEGF
vascular endothelial growth factor
vascular endothelial growth factor


NM_007040
2.432915688
E1B-AP5
E1B-55 kDa-associated protein 5
E1B-55 kDa-associated protein 5






isoform a; E1B-55 kDa-associated






protein 5 isoform d; E1B-55 kDa-






associated protein 5 isoform b;






E1B-55 kDa-associated protein 5






isoform c


NM_001402
2.404632807
EEF1A1
eukaryotic translation elongation
eukaryotic translation elongation





factor 1 alpha 1
factor 1 alpha 1


NM_002357
2.396934271
MAD
MAX dimerization protein 1
MAX dimerization protein 1


NM_005524
2.39485383
HES1
hairy and enhancer of split 1,
hairy and enhancer of split 1





(Drosophila)


NM_005730
2.387011766
CTDSP2
conserved gene amplified in
nuclear LIM interactor-interacting





osteosarcoma
factor 2


AF040963
2.356589317
MXD4
MAX dimerization protein 4
MAD4


M83221
2.351067066
RELB
v-rel reticuloendotheliosis viral
reticuloendotheliosis viral





oncogene homolog B, nuclear factor
oncogene homolog B





of kappa light polypeptide gene





enhancer in B-cells 3 (avian)


NM_006842
2.24469161

splicing factor 3b, subunit 2, 145
interleukin-1 receptor-associated





kDa
kinase 2


NM_001570
2.239902258
IRAK2
interleukin-1 receptor-associated
activator of S phase kinase





kinase 2


NM_006716
2.230443954
ASK
activator of S phase kinase
rho/rac guanine nucleotide






exchange factor 2


NM_004723
2.209302187
ARHGEF2
rho/rac guanine nucleotide exchange
human immunodeficiency virus type I





factor (GEF) 2
enhancer binding protein 2


M60119
2.204995632
HIVEP2; MBP-2;
human immunodeficiency virus type
ras homolog gene family, member G




HIV-EP2
I enhancer binding protein 2
(rho G)


NM_001665
2.111301422
ARHG
ras homolog gene family, member G
early growth response 1





(rho G)


M62829
2.104651213
EGR1
early growth response 1
RAB11B, member RAS oncogene family


NM_004218
2.102017164
RAB11B
RAB11B, member RAS oncogene family


NM_006318
2.098191261

putative glialblastoma cell
MYC-associated zinc finger protein





differentiation-related


U33819
2.0930233
MAZ
MYC-associated zinc finger protein





(purine-binding transcription





factor)


M13150
2.069767475
MAS1
MAS1 oncogene
MAS1 oncogene


NM_003685
2.039983511
KHSRP
KH-type splicing regulatory protein
KH-type splicing regulatory protein





(FUSE binding protein 2)
(FUSE binding protein 2)


NM_006694
2.018802404
JTB; PAR; hJT;
jumping translocation breakpoint
jumping translocation breakpoint




HJTB; B PAR


NM_006191
2.002365112
PA2G4
proliferation-associated 2G4, 38
proliferation-associated 2G4, 38





kDa
kDa


X78710
2.001409531
MTF1
metal-regulatory transcription
metal-regulatory transcription





factor 1
factor 1















Gene Name
Phenotype
Function
Keywords







K03218
Colon cancer, advanced

c-myc proto-oncogene; proto-






oncogene; src gene; src oncogene



NM_001201



D16431


hepatoma-derived GF; hepatoma-






derived growth factor



X54936


placenta growth factor



M95712
Adenocarcinoma of lung,

b-raf oncogene; serine/threonine




somatic; Colorectal cancer,

protein kinase




somatic; Melanoma,




melignant, somatic;




Nonsmall cell lung cancer,




somatic



NM_005850



NM_005937



NM_001419



NM_002613

protein kinase



NM_014308



D26120



X61498


NF-kb subunit



X03663
Myeloid malignancy,

c-fms oncogene; fms oncogene;




predisposition to

glycoprotein; membrane protein;






proto-oncogene; signal peptide



NM_004204



NM_003017



NM_000753



NM_002712

regulatory polypeptide of





protein phosphatase-1



NM_004906



NM_005207



NM_007279



NM_015714



NM_001511



NM_016263



NM_012103



M32977
Diabetic retinopathy,

angiogenic mitogen; vascular




NIDDM-related,

endothelial growth factor




susceptibility to



NM_007040



NM_001402



NM_002357



NM_005524



NM_005730



AF040963



M83221

NF-kappa-B transcpription
I-Rel; NF-kappa-B transcription





factor p50-subunit inhibitor
factor inhibitor



NM_006842



NM_001570



NM_006716



NM_004723



M60119



NM_001665



M62829


transcription factor



NM_004218



NM_006318



U33819



M13150


mas oncogene; mas protein;






membrane protein; proto-oncogene



NM_003685



NM_006694



NM_006191



X78710


metal-regulatory transcription






factor; MTF-1 gene; transcription






factor

















TABLE 2





Immune system gene regulated by the HPLC-purified peptide


Immune system gene enhanced by the peptide




















Gene Name
TDP
Description
Product















1
K02770
11.69
interleukin 1, beta
interleukin 1, beta proprotein


2
J04130
7.341
chemokine (C—C motif) ligand 4
chemokine (C—C motif) ligand 4 precursor


3
NM_000211
3.326
integrin, beta 2 (antigen CD18 (p95),
integrin beta chain, beta 2 precursor





lymphocyte function-associated antigen





1; macrophage antigen 1 (mac-1) beta





subunit)


4
X03663
2.781
colony stimulating factor 1 receptor,
colony stimulating factor 1 receptor





formerly McDonough feline sarcoma
precursor





viral (v-fms) oncogene homolog


5
NM_006186
2.06
nuclear receptor subfamily 4, group A,
nuclear receptor subfamily 4, group A,





member 2
member 2 isoform a; nuclear receptor






subfamily 4, group A, member 2 isoform






b; nuclear receptor subfamily 4, group A,






member 2 isoform c; nuclear receptor






subfamily 4, group A, member 2 isoform d


6
NM_002000
1.871
Fc fragment of IgA, receptor for
Fc alpha receptor isoform a precursor;






Fc alpha receptor isoform b precursor;






Fc alpha receptor isoform c precursor;






Fc alpha receptor isoform d; Fc alpha






receptor isoform e; Fc alpha receptor






isoform f; Fc alpha receptor isoform g;






Fc alpha receptor isof


7
NM_003199
1.621
transcription factor 4
transcription factor 4 isoform b


8
NM_001733
1.543
complement component 1, r subcomponent
complement component 1, r subcomponent


9
NM_002983
1.542
chemokine (C—C motif) ligand 3
chemokine (C—C motif) ligand 3


10
NM_002969
1.53
mitogen-activated protein kinase 12
mitogen-activated protein kinase 12


11
NM_000173
1.529
glycoprotein Ib (platelet), alpha
platelet glycoprotein Ib alpha





polypeptide
polypeptide precursor


12
D10202
1.506
platelet-activating factor receptor
platelet-activating factor receptor


1
NM_006864
0.662
leukocyte immunoglobulin-like receptor,
leukocyte immunoglobulin- like receptor,





subfamily B (with TM and ITIM
subfamily B (with TM and ITIM





domains), member 3
domains), member 3


2
NM_007052
0.662
NADPH oxidase 1
NADPH oxidase 1 isoform long; NADPH






oxidase 1 isoform short; NADPH






oxidase 1 isoform long variant


3
NM_002258
0.653
killer cell lectin-like receptor
killer cell lectin-like receptor





subfamily B, member 1
subfamily B, member 1


4
NM_001776
0.646
ectonucleoside triphosphate
ectonucleoside triphosphate





diphosphohydrolase 1
diphosphohydrolase 1


5
NM_016523
0.644
killer cell lectin-like receptor
killer cell lectin-like receptor





subfamily F, member 1
subfamily F, member 1


6
NM_014442
0.641
sialic acid binding Ig-like lectin 8
sialic acid binding Ig-like lectin 8


7
NM_004133
0.635
hepatocyte nuclear factor 4, gamma
hepatocyte nuclear factor 4, gamma


8
NM_002121
0.633
major histocompatibility complex class
major histocompatibility complex, class





II, DP beta 1
II, DP beta 1 precursor


9
NM_000397
0.631
cytochrome b-245, beta polypeptide
cytochrome b-245, beta polypeptide





(chronic granulomatous disease)
(chronic granulomatous disease)


10
NM_004750
0.626
cytokine receptor-like factor 1
cytokine receptor-like factor 1


11
NM_003891
0.618
protein 2, vitamin K-dependent plasma
protein Z, vitamin K-dependent plasma





glycoprotein
glycoprotein


12
NM_002260
0.603
killer cell lectin-like receptor
killer cell lectin-like receptor





subfamily C, member 2
subfamily C, member 2


13
NM_005545
0.579
immunoglobulin superfamily containing
immunoglobulin superfamily containing





leucine-rich repeat
leucine-rich repeat


14
NM_001311
0.561
cysteine-rich protein 1 (intestinal)
cysteine-rich protein 1 (intestinal)


15
NM_004528
0.561
microsomal glutathione S-transferase 3
microsomal glutathione S-transferase 3


16
NM_018661
0.528
defensin, beta 103
defensin, beta 103, precursor


17
NM_002124
0.502
major histocompatibility complex class
major histocompatibility complex, class





II, DR beta 1
II, DR beta 1 precursor


18
Y00815
0.475
protein tyrosine phosphatase,
protein tyrosine phosphatase,





receptor type, F
receptor type, F isoform 1 precursor;






protein tyrosine phosphatase, receptor






type, F isoform 2 precursor


19
NM_004636
0.459
sema domain, immunoglobulin domain
sema domain, immunoglobulin domain





(Ig), short basic domain, secreted,
(Ig), short basic domain, secreted,





(semaphorin) 3B
(semaphorin) 3B


20
NM_001783
0.4
CD79A antigen (immunoglobulin-
CD79A antigen isoform 1 precursor;





associated alpha)
CD79A antigen isoform 2 precursor


21
NM_004106
0.382
Fc fragment of IgE, high affinity I,
Fc fragment of IgE, high affinity I,





receptor for; gamma polypeptide
receptor for, gamma polypeptide






precursor


22
U77604
0.349
microsomal glutathione S-transferase 2
microsomal glutathione S-transferase 2


23
NM_002118
0.335
major histocompatibility complex,
major histocompatibility complex,





class II, DM beta
class II, DM beta precursor


24
NM_002123
0.328
major histocompatibility complex,
major histocompatibility complex,





class II, DQ beta 1
class II, DQ beta 1 precursor


25
K01171
0.319
major histocompatibility complex,
major histocompatibility complex,





class II, DR alpha
class II, DR alpha precursor


26
NM_006120
0.298
major histocompatibility complex,
major histocompatibility complex,





class II, DM alpha
class II, DM alpha precursor















Gene Name
Phenotype
function
Keywords

















1
K02770

interleukin-1 receptor binding;
interleukin; interleukin 1






signal transducer activity



2
J04130

receptor signaling protein
act2 gene; immune activation






tyrosine kinase activity;
gene






chemokine activity



3
NM_000211
Leukocyte adhesion
cell adhesion receptor activity





deficiency



4
X03663
Myeloid malignancy,
macrophage colony stimulating
c-fms oncogene; fms oncogene;





predisposition to
factor receptor activity; ATP
glycoprotein; membrane protein;






binding; transferase activity
proto-oncogene; signal peptide



5
NM_006186
Parkinson disease
steroid hormone receptor






activity; transcription factor






activity



6
NM_002000

receptor activity; receptor






signaling protein activity



7
NM_003199

RNA polymerase II transcription






factor activity; DNA binding



8
NM_001733
Clr/Cls deficiency,
complement component Clr





combined
activity;trypsin activity;






calcium ion binding;






chymotrypsin activity;






hydrolase activity



9
NM_002983

chemokine activity; antiviral






response protein activity;






signal transducer activity



10
NM_002969

MAP kinase activity; ATP






binding; protein serine/






threonine kinase activity;






transferase activity; SAP






kinase 3 activity



11
NM_000173
Bernard-Soulier
thrombin receptor activity;





syndrome
cell adhesion molecule activity



12
D10202

platelet activating factor
G-protein coupled receptor; PAF






receptor activity
receptor; platelet-activating







factor receptor



1
NM_006864

receptor activity



2
NM_007052

superoxide-generating NADPH






oxidase activity; oxidoreductase






activity; voltage-gated proton






channel activity



3
NM_002258

sugar binding; transmembrane






receptor activity



4
NM_001776

apyrase activity; magnesium ion






binding; hydrolase activity



5
NM_016523

transmembrane receptor activity



6
NM_014442

sugar binding; transmembrane






receptor activity; cell adhesion






molecule activity



7
NM_004133

steroid hormone receptor






activity; steroid binding;






transcription factor activity



8
NM_002121
Beryllium disease,
class II major histo-





chronic,
compatibility complex antigen





susceptibility to



9
NM_000397
Chronic
voltage-gated ion channel





granulomatous
activity; electron transporter





disease, X-linked
activity; oxidoreductase






activity



10
NM_004750
Cold-induced
receptor activity





sweating syndrome



11
NM_003891

trypsin activity; protein






binding; calcium ion binding;






chymotrypsin activity



12
NM_002260

sugar binding; transmembrane






receptor activity



13
NM_005545

protein binding



14
NM_001311

zinc ion binding



15
NM_004528

peroxidase activity; glutathione






transferase activity



16
NM_018661

antimicrobial peptide activity;






Gram-positive antibacterial






peptide activity



17
NM_002124
Pemphigoid,
MHC class II receptor activity





susceptibility to



18
Y00815


antigen; cell surface







glycoprotein; glycoprotein;







immunoglobulin superfamily; LAR







gene; leukocyte common antigen;







neural cell adhesion molecule;







transmembrane protein



19
NM_004636



20
NM_001783

transmembrane receptor activity



21
NM_004106

receptor signaling protein






activity; transmembrane






receptor activity; IgE binding



22
U77604

glutathione transferase






activity; enzyme activator






activity



23
NM_002118

chaperone activity; MHC class






II receptor activity



24
NM_002123
Creutzfeldt-Jakob





disease, variant,





resistance to



25
K01171

MHC class II receptor activity
antigen; class II antigen;







histocompatibility antigen;







major histocompatibility complex



26
NM_006120










The present inventor has verified the validity of this diagnosis method using 55000 cancer-related genes. When such a great number of cancer-related genes are used, DNA chip technology is preferably used. In the DNA chip technology, each DNA of the cancer-related genes is immobilized so as to be arrayed on a slide; the above-mentioned labeled cRNA solution is dropped into the slide; the cRNA which is not hybridized is removed by washing; and fluorescence intensity of each gene position is detected by a scanner. The results are compared with the result which is obtained by conducting the same process using the above-mentioned control solution. Thus, cancer-related genes of which expression is increased or decreased when cells or tissues are treated with the peptide of the present invention can be exhaustively analyzed.
Therefore, a combination of such a cancer-related gene, the peptide of the present invention, and a polynucleotide encoding the peptide is useful as a cancer diagnostic reagent kit for determining a tendency of canceration or malignancy of cancer.
[Diagnosis Method 3]
The peptide of the present invention is also used as an antigen for preparing a specific antibody against the peptide. The specific antibody is useful as a diagnostic reagent. The antibody may be either polyclonal or monoclonal antibody, and these antibodies can be prepared by known methods. For example, the polyclonal antibody can be prepared as an antiserum by immunizing an animal such as mouse, rat, or rabbit with the peptide of the present invention. The antiserum may be further purified. The monoclonal antibody can be prepared by a hybridoma method: extracting spleen cells from the above-mentioned sensitized animal and fusing the spleen cells with myeloma cells to form hybridomas.
In the immunization, an appropriate adjuvant may be used. In addition, the peptide of the present invention may be used as a conjugate with a carrier protein such as keyhole limpet hemocyanin for increasing the immunogenicity of the peptide.
The antibody against the peptide of the present invention is used as a diagnostic reagent for determining malignancy of cancer, wherein a specimen, such as cancer tissues or cells, is stained by using the specific antibody labeled with an appropriate fluorescence dye or the like. When the peptide of the present invention is detected in the tissues or cells, it is suggested that cancer is advancing. The amount of the peptide can be determined by measuring the fluorescence intensity. A large amount of the peptide indicates that the malignancy of cancer is considerably high.
Additionally, the antibody against the peptide of the present invention can be used as a diagnostic reagent for measuring a tendency of canceration (easiness of conversion to cancer: a degree of risk of canceration). In such a use of the antibody, a specimen of the tissues or cells shown in diagnosis methods 1 and 2 is stained using the labeled antibody. When the peptide of the present invention is detected in these tissues or cells, it is suggested that the tissues or cells are exposed to cancer-inducing stress, i.e., a degree of risk of canceration is high. When the amount of the peptide of the present invention is large, it is suggested that the tendency is further higher. When the specimen is cancer cells, a large amount of the peptide suggests that the malignancy of the cancer is high.
Furthermore, the antibody against the peptide of the present invention can be expected to have an anticancer effect such as suppression of cancer cell proliferation as a cancer-cell proliferation-suppressing drug. In addition, the antibody can be expected to block malignant alteration or mutation acceleration to which normal cells are subjected as a cancer-preventing drug. Furthermore, by binding the antibody to an anticancer agent, a drug which may be used for missile therapy can be obtained.
EXAMPLES
Examples of the present invention will now be described, but the present invention is not limited to these examples.
Example 1
Preparation and Sequencing of the Peptide of the Present Invention
(Cell: UTC-8)
The squamous-cell carcinoma cell (UTC-8: FERM BP-08611) is a highly differentiated type with high metastasis potential and keratinization tendency. The cell also has high adhesion and proliferation ability and becomes confluent in 5 to 7 days after inoculating 1×105 cells into a culture medium (5 ml) in a T25 culture flask (Falcon).
(Cell-culturing condition)
The following processes were performed: inoculating 1×106 UTC-8 cells into 10 T150 culture flasks (Falcon); performing initial culture in 20 ml RPMI1640 culture medium with 10% FCS (GIBCO); and additionally supplying 30 ml of the culture medium when the proliferation reached about 30% of the culture flask dimension. The culture was performed in 5% CO2 and 95% humidity at an incubator internal temperature of 37° C. When the cells become 80% confluent (the proliferation of the cells is maintained at the point just before the cell proliferation reaches its peak), the antigen peptide bound to HLA was eluted from the cell membrane surfaces.
(Antigen Peptide-eluting Procedure)
The culture medium for the UTC-8 cells was removed when the cells became 80% confluent in each T150 culture flask (Falcon). The cells were washed with Hanks solution once and then with 30 ml of a PBS solution not containing divalent calcium ion and divalent magnesium ion twice. Then, after the sufficient removal of the solution, 10 ml of a citrate-phosphate buffer having a pH of 3.3 to 3.4 was added to each flask, and the cells were left at a room temperature for 2 min.
Then, the citrate-phosphate buffer solution was collected and centrifuged at 1200 rpm for 7 min, and the supernatant was filtered through a 0.45 μm filter (Millex-HV PVDF: MILLIPORE). The filtrate was further filtered through a 0.22 μm filter. The filtrate was desalted with Sep-Pac C18 cartridge (Waters), and the target substance bound to the cartridge was eluted with a 60% (v/v) acetonitrile aqueous solution as a crude extract solution. The thus obtained crude extract solution was frozen and stored at −20° C. for later HPLC.
The cells received the above-described treatment were washed, immediately after the collection with the citrate-phosphate buffer solution having a pH of 3.3 to 3.4, with 30 ml of Hanks solution twice. Then, the cells were recultured in 30 ml of RPMI 164 medium containing 10% FCS. This treatment was subjected to the same cells once a day for successive 4 days.
(Lyophilization and Redissolution of Crude Extraction)
The crude extract solution obtained in above was lyophilized using a lyophilizer (FD-1000: EYELA) under conditions at a trapping temperature of −40° C. and at a degree of vacuum of 15 Pa or less. The dried sample was redissolved in 5 ml of the citrate-phosphate buffer solution having a pH of 3.3 to 3.4. At this stage, the total protein amount determined by Lowry method was 400 μg.
(Two-Dimensional Electrophoresis)
Ten micrograms of the thus obtained molecule was applied to two-dimensional electrophoresis, but no band was visible to the naked eye by Coomassie Brilliant Blue (CBB) R250 staining (detection sensitivity: 1 μg) and also by silver staining having a sensitivity as high as about 1000 times that of the CBB staining, (theoretical sensitivity: 1 ng, actual sensitivity: about 20 times that of the CBB staining). This suggests that the peptide molecule physically passes through a gel (silica gel C18) generally used in the two-dimensional electrophoresis. This is an unusual characteristic of the peptide molecule of the present invention.
(Fractionating by Gel Filtration)
Since the molecule of the present invention cannot be detected by usual two-dimensional electrophoresis, it was tried to detect the molecule by an HPLC system. The target peptide was obtained as a fraction obtained by using an AKTA Explorer 10 (Amersham Pharmacia Biotech) HPLC system and Superdex Peptide 10/300 GL (Amersham Pharmacia Biotech) as a gel-filtration column; using a citrate-phosphate buffer solution (0.1 M citric acid and 0.2 M phosphoric acid) having a pH of 6.8 as an elution buffer at a flow rate of 0.2 ml/min; and isolating a peak in a fraction at an elution volume of 20.8 to 22.8 ml by using a detection wavelength of 214 nm. The obtained fraction was lyophilized and then redissolved in sterilized redistilled water for desalting. The desalting was performed by fractionating by the AKTA Explorer 10 (Amersham Pharmacia Biotech) HPLC system and Superdex Peptide 10/300 GL (Amersham Pharmacia Biotech) as a gel-filtration column using distilled water as a solvent at a flow rate of 0.2 ml/min. The target peptide was obtained as a fraction at an elution volume of 11.4 to 20.0 ml.
FIG. 1 shows an elution profile of the gel filtration.
The peptide was lyophilized using the lyophilizer FD-1000 (EYELA) and stored at −20° C. for later mass spectrometry.
(Optimal pH)
The optimal pH of the elution buffer was 6.8 to 7.2. When the pH of the elution buffer was 7.3 or more, peaks were further divided into smaller peaks. When the pH of the elution buffer was 6.7 or less, peaks, which were isolated from each other at a pH of 6.8, lapped over each other, and the gel filtration resolution was decreased.
(Detection Wavelength)
The peptide is characterized by the fact that the peak of the peptide in the crude extract solution can be separately detected by using physiological activity as an indicator only when a detection wavelength of 214 nm is used. However, the peptide cannot be detected when a detection wavelength of 254 to 257 nm or of 280 nm, which are generally used in peptide detection, is used. These results suggest that the amino acid composition of the peptide has extremely low contents of tyrosine (absorption wavelength: 280 nm), tryptophan (absorption wavelength: 280 nm), and phenylalanine (absorption wavelength: 257 nm); or that the peptide does not substantially contain such amino acids. The use of a detection wavelength of 214 nm has a demerit such that many interfering substances are also detected by ultraviolet absorption spectrometry. However, it can precisely measure a protein amount (quantitative detection range: 5 to 1000 μg) regardless of types of proteins. The 214 nm is the most appropriate wavelength for, as in this experiment, precisely measuring a concentration of unknown peptide in a crude extract solution by detecting a peak utilizing peptide-bond absorption without denaturation of the peptide to maintain the physiological activity. On the other hand, the quantitative detection property was further improved by using a detection wavelength of 205 nm when the crude extract solution containing the peptide was applied to a desalting column (Sep-Pack 18) or to a molecular sieve (Centricon YM-3) for removing foreign substances such as nucleic acids as far as possible, by intending only purification of the peptide without any concern for maintaining the physiological activity. Therefore, when a peak in the crude extract solution was detected using the physiological activity as an indicator, a detection wavelength of 214 nm was used, and when the precise quantitative determination was necessary, a detection wavelength of 205 nm was used.
(Fractionating Flow Rate in Column)
The fractionating flow rate is important for separating the peptide. Namely, the flow rate of an elution buffer is determined so that molecules slowly pass through the column to effectively utilize advantages of the molecular sieve. When the flow rate was 0.28 ml/min, adjacent peaks lapped over each other; thus, the separation resolution was insufficient. For the detection of the peak of the present invention, a low flow rate of 0.14 ml/min was optimal. Each fraction was determined to be 0.1 ml/well from the view point of the width of the peak, and was collected in a 96-well plate (Nunc).

(Structure Analysis of the Peptide by Mass Spectrometer)

The peptide fraction that was stored after lyophilization was redissolved in sterilized redistilled water, and 5 μg as a protein was separated by ProteinChip Series 4000 system (Ciphergen). The sample (5 μg) was bound to the ProteinChip by using MilliQ water as a binding/wash buffer in normal-phase ProteinChip and using 100 mM sodium acetate (pH 4.0) as a binding/wash buffer in cation exchange ProteinChip, and was measured by using alpha-cyano-4-hydroxy-cinnamic acid (CHCA) as an energy-absorbing molecular in a mass range of 800 to 2500 m/z. As a result, a peak of a molecular weight which was thought to be that of the target peptide was detected. By using the detection conditions clarified by the above-described processes as a reference, the target peptide trapped on the chip was analyzed by a mass spectrometer, QSTAR XL LC/MC/MS system (Applied Biosystems). Then, on the basis of the results of time-of-flight (TOF mass spectrometry) analysis of the fragmented peptide-constituting portion, a data base (Mascot Search Results: Matrix Science) was searched for a sequence of the peptide to reveal that the peptide had the following peptide sequence of which function was unknown as of Jan. 27, 2005.









Peptide:




Gln-Pro-Gln-Phe-Gly-Arg-Arg-Met-Glu-
(SEQ ID NO: 1)


Ser-Lys






Example 2
Confirmation of Expression of the Peptide of the Present Invention in Original Cancer Cell UTC-8
The fact that the peptide obtained in Example 1 was actually expressed in the original cancer cell UTC-8 at a gene level was confirmed as follows:
UTC-8 cells after the extraction of the peptide of the present invention were recultured, and total RNA was extracted from the recultured UTC-8 cells after 2 hr, 4 hr, and 6 hr from the extraction, respectively, by using an RNA extraction kit (QIAGEN). Then, cDNA was synthesized using each of the extracted total RNA as a template to produce a cDNA pool. On the basis of the amino acid sequence (SEQ ID NO: 1) of the peptide, the following primers were designed. Fw primers (DNAs encoding four contiguous amino acids of the peptide were synthesized so that the N-terminal of the four contiguous amino acids shifted one by one from the N-terminal of the peptide toward the C-terminal.)












MHC 1-4
CAR CCN CAR TTY
(SEQ ID NO: 2)




 



MHC 2-5
CCN CAR TTY GGN
(SEQ ID NO: 3)



 



MHC 3-6
CAR TTY GGN AGR
(SEQ ID NO: 4)



 



MHC 4-7
TTY GGN AGR AGR
(SEQ ID NO: 5)



 



MHC 5-8
GGN AGR AGR ATG
(SEQ ID NO: 6)



 



MHC 6-9
AGR AGR ATG GAR
(SEQ ID NO: 7)



 



MHC 7-10
AGR ATG GAR TCN
(SEQ ID NO: 8)



 



MHC 8-11
ATG GAR TCN AAR
(SEQ ID NO: 9)






(N: A, T, C, or G, R: G or A, Y: C or T) Rv primer (a gene sequence produced for an amino acid sequence at the C-terminal of the peptide): Oligo-dT primer (poly-A primer)

Here, the poly-A primer was used as the Rv primer in order to obtain cDNA synthesized from mRNA.
Complementary DNAs were synthesized by reverse transcription from total RNA which was extracted at each time to produce a cDNA pool. Then, PCR was performed using this cDNA pool as templates and using primers synthesized as described above under conditions at 94° C. for 3 min, at 55° C. for 1 min, and at 74° C. for 1 min as one cycle. The cycle was repeated 35 cycles in total. After agarose-gel electrophoresis, 12 bands were selected from a sample of which RNA was extracted after 4 hr when the signal intensity was largest. The size of each band was about 500 to 2000 bp. Genes of these bands extracted from the gel were inserted into pGM easy vectors.
Insert of the genes into 12 types of the pGM easy vectors was checked, and 8 samples per one type of the vector were sequenced. As a result, it was confirmed that all the sequences of the 8 samples included a common sequence shown in Table 3 below, though the DNA lengths of the sequences were different from those of each other.







TABLE 3




















The result shows the fact that a gene being common in or complementary to the gene encoding the peptide of the present invention is surely present in original cancer cell UTC-8 and that the peptide is derived from UTC-8 cells.
Example 3
Artificial Synthesis of the Peptide of the Present Invention by Fmoc Method
The peptide was artificially synthesized by an in vitro synthesis system (Abacus: Sigma Genosys).
The peptide was synthesized using an activated cellulose membrane (SPOTs: Sigma Genosys) as follows: The α-amino groups of amino acids were protected with 9-fluorenyl methoxy carbonyl (Fmoc) and the carboxyl groups were protected with an active ester (Opfp or Odhbt). These amino acid derivatives were dissolved in 1-methyl-2-pyrrolidinone (NMP). About 1 μl of the amino acid derivative solution (equivalent to 100 to 200 μg of Fmoc-amino acid) was spotted on the membrane at each marked position where a spacer arm was bound thereto. The end of the spacer arm had a free amino group, and the spots were colored to blue with bromophenol blue (BPB). An amido bond was formed by the reaction between the amino group on the membrane and the active ester. Then, capping was carried out as follows: Excess amino acid derivatives were rinsed away with dimethylformamide (DMF), and then unreacted amino group was acetylated by treating with acetic anhydride/DMF so as to be lost the reactivity. Then, the Fmoc group protecting the amino group was removed by secondary amine, piperidine/DMF, in order to let the amino group free for the subsequent extension reaction. Additionally, after all synthesis processes were completed, deprotection of the side chains was carried out. Namely, a t-butyl alcohol-based protecting group (Pmc, OtBu, Trt, tBoc, tBu, etc.) protecting the reactive side chains was removed by using trifluoroacetate (TFA) mixed with dichloromethane (DCM)/triisobutylsilane. Thus, the target peptide was artificially synthesized.
Example 4
Influence of the Artificially Synthesized Peptide on Normal Cell Gene
(a) The peptide obtained in Example 3 was added to a human normal peripheral blood monocyte culture system (10 to 100 μg peptide/1×106 to 1×107 monocytes), and they were cocultured for 7 hr. Then, mRNA was extracted from the monocytes and was applied to analysis for up-regulation and down-regulation in respect to about 55000 human genes by using DNA chip system (Clontech). Table 4 shows a part of the genes.







TABLE 4







A. Enhanced Gene


















Expression Amount
Expression Amount



Gene



before Treatment
after Treatment with



No.
Gene Name
Gene Bank

with the Peptide
the Peptide





1. Oncogene








1
21713
NM_004339.2
GE480864
DISCOVERY
0.168670654
7.741943359


2
32050
CB999164.1
GE540894
DISCOVERY
0.344818115
9.805541992


3
34854
NM_198502.1
GE603511
DISCOVERY
0.040802002
1.098205566


4
46997
AL831827.1
GE547122
DISCOVERY
11.17044067
236.1818237


5
37781
AI478531.1
GE645872
DISCOVERY
2.533325195
32.40939331


6
7394
AA021565.1
GE707587
DISCOVERY
29.597229
350.270813


7
22388
CD244420.1
GE692174
DISCOVERY
60.32098389
571.2325439


8
6231
BF973345.1
GE54360
DISCOVERY
0.583343506
5.453491211


9
34979
BC041456.1
GE631189
DISCOVERY
2.651672363
24.171875


10
4210
NM_152577.1
GE493259
DISCOVERY
61.90475464
393.7416382


11
36631
AW302705.1
GE53805
DISCOVERY
71.11248779
417.5


12
29317
AA60U91.1
GE551636
DISCOVERY
2.891571045
10.05682373


13
14195
NM_002170.2
GE55306
DISCOVERY
2.811950684
7.21875


14
2183
AA861218.1
GE80525
DISCOVERY
3.408691406
8.622650146


15
22615
BX117642.1
GE516913
DISCOVERY
81.63721924
204.4117737


16
3858
AI263196.1
GE59314
DISCOVERY
9.184204102
22.61538696


17
8431
NM_021081.3
GE56485
DISCOVERY
7.641021729
18.609375


18
18829
NM_002958.1
GE81500
DISCOVERY
6.883331299
15.3151959


19
8440
NM_016232.4
GE79419
DISCOVERY
7.225799561
15.71429443


20
3642
AW594132.1
GE543964
DISCOVERY
1627.052002
3306.942871


21
38566
NM_031273.1
GEB4875
DISCOVERY
7.339630127
14.54998779


22
24910
NM_018556.2
GE60436
DISCOVERY
29.01333618
55.05334473


23
6329
BX116538.1
GE58014
DISCOVERY
37.80822754
67.53659058


2. Immune


System Gene


1
25767
INCYTE UNIQUE
GE59636
DISCOVERY
130.6451721
4927.383301


2
29703
BX103139.1
GE59980
DISCOVERY
0.660003662
19.8302002


3
28615
AA504638.1
GE520093
DISCOVERY
2.739135742
20.69332886


4
30707
AI809890.1
GE54001
DISCOVERY
2.853668213
21.05661011


5
21607
NM_002960.1
GE80951
DISCOVERY
3.446289063
24.92727661


6
51904
H08511.1
GE893705
DISCOVERY
2.304870605
11.3684082


7
22254
NM_176891.2
GE80659
DISCOVERY
26.98571777
122.6947327


8
45170
BM671692.1
GE53155
DISCOVERY
38.31506348
85.25881958


9
22100
BX457477.2
GE616415
DISCOVERY
328.440918
608.4909058










A. Enhanced Gene










Enhancement




Ratio
Description





1. Oncogene


1
 45.89976479
zn87blly5 Stratagene lung carcinoma 937218 cDNA clone IMAGE: 565149 5′ similar to




contains Alu repetitive element; contains element MER22 repetitive element;


2
 28.43685282
disrupted in renal carcinoma 1 (DIRC1)


3
 26.91548242
Homo sapiens NEUROBLASTOMA COT 25-NORMALIZED cDNA clone




CS0DC018YE24 3-PRIME


4
 21.14346521
clone N11 NTera2D1 teratocarcinoma mRNA


5
 12.79322267
NEUROBLASTOMA COT 25-NORMALIZED cDNA clone CS0DC011YD03 5-PRIME


6
 11.8345813
HELA CELLS COT 25-NORMALIZED cDNA clone CS0K007YM05 3-PRIME


1
 9.469881078
zb91f05s1 Soares_parathyroid_tumor_NbHPA cDNA clone IMAGE: 320193 3′


8
 9.348679048
breast carcinoma amplified sequence 1 (BCAS1)


9
 9.115709518
yu38e06r1 Soares ovary tumor NbHOT cDNA clone IMAGE: 236098 5′


10
 6.360442594
RAS-like, family 11, member A (RASL11A)


11
 5.87098009
RAB43, member RAS oncogene family (RAB43), mRNA


12
 3.477979124
T-cell leukemia/lymphoma 6 (TCL6), transcript variant TCL6a1


13
 2.567168067
B-cell translocation gene 4 (BTG4)


14
 2.529607148
neuro-oncological ventral antigen 1 (NOVA1), transcript variant 1


15
 2.497784939
endogenous retroviral family W, env(C7), member 1 (syncytin) (ERVWE1), mRNA


16
 2.46242208
preferentially expressed antigen in melanoma (PRAME), transcript variant 3


17
 2.435456365
Ras-associated protein Rap1 (RBJ)


18
 2.225055752
v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian)




(MYCN)


19
 2.174748179
mab-21-like 2 (C elegans) (MAB21L2)


20
 2.032475217
RAB2, member RAS oncogene family (RAB2)


21
 1.982387061
RAS protein activator like 2, mRNA (cDNA clone IMAGE: 5399841), with apparent




retained intron


22
 1.891518589
platelet-derived growth factor beta polypeptide (simian sarcoma viral (v-sis)




oncogene homolog) (PDGFB), transcript variant 2


23
 1.186293486
mutated in colorectal cancers (MCC)


2. Immune


System Gene


1
 37.71577029
tumor necrosis factor (TNF superfamily, member 2) (TNF)


2
 30.04559116
chemokine (C-X-C motif) ligand 9 (CXCL9)


3
 7.554692722
IL3-UT0117-080301-496-D07 UT0117 Homo sapiens cDNA


4
 7.378787069
immunoglobulin superfamily, member 4C (IGSF4C)


5
 7.233077713
colony stimulating factor 1 (macrophage) (CSF1), transcript variant 1


6
 4.932341181
interleukin 1 family, member 8 (eta) (IL1F8), transcript variant 1


7
 4.546654408
interleukin 4 (IL4), transcript variant 1


8
 2.225203663
transforming growth factor beta 1 induced transcript 1 (TGFB1I1)


9
 1.852664734
IL5-CI0001-181000-178-f03 CI0001 Homo sapiens cDNA










B. Suppressed Gene


















Expression Amount
Expression Amount



Gene



before Treatment
after Treatment with



No.
Gene Name
Gene Bank

with the Peptide
the Peptide





1. Cancer-








Suppressing








Gene


1
35687
BX108016.1
GE82100
DISCOVERY
103.9224243
0.225219727


2
36334
BF433017.1
GE82101
DISCOVERY
341.440918
2.079650879


3
37600
AW979088.1
GE62125
DISCOVERY
982.9432373
8.42855835


4
30909
NM_018412.2
GE79080
DISCOVERY
987.7108154
11.47369385


5
8280
BC063301.1
GE62103
DISCOVERY
758.4154053
9


6
11436
NM_152271.2
GE82056
DISCOVERY
1207.091431
14.74417114


7
11261
NM_018559.2
GE56225
DISCOVERY
379.1338501
6.257141113


8
50395
NM_004480.3
GE79552
DISCOVERY
470.2086792
8.763153076


9
52111
AK056875.1
GE61359
DISCOVERY
593.6870728
13.6000061


2. Immune


System Gene


1
20881
NM_001482.1
GE61110
DISCOVERY
6599.050293
38.16882324


2
47973
NM_004394.1
GE81458
DISCOVERY
1480.68396
9.638885498


3
10791
W00901.1
GE80378
DISCOVERY
517.4453125
3.42855835


4
15907
BU899259.1
GE80960
DISCOVERY
912.8641357
6.363647461


5
16756
AI707455.1
GE81522
DISCOVERY
395.0359497
2.80645752


6
34736
AV736303.1
GE80100
DISCOVERY
3974.221191
29.0786438


7
39991
AI252940.1
GE61199
DISCOVERY
2414.748779
17.77420044


8
41726
AI453596.1
GE58507
DISCOVERY
4512.05127
33.50683594


9
45767
BG190549.1
GE79374
DISCOVERY
2359.263184
18.125


10
42614
AW296107.1
GE81744
DISCOVERY
626.2438965
4.848480225


11
20551
AK024566.1
GE86210
DISCOVERY
2811.726807
22.93103027


12
38103
NM_173695.1
GE61298
DISCOVERY
703.8875122
5.777770996


13
37068
AW971488.1
GE58019
DISCOVERY
6960.161133
57.38461304


14
4899
BX115183.1
GE80961
DISCOVERY
536.4679565
4.5


15
43032
BC013284.2
GE58028
DISCOVERY
519.3710938
4.445770264


16
42813
NM_001763.1
GE58393
DISCOVERY
4447.713379
38.51351929


17
22326
NM_014396.2
GE82907
DISCOVERY
1102.541626
9.742858887


18
37445
NM_007068.2
GE60016
DISCOVERY
1671.436768
14.875


19
22890
BX119833.1
GE86305
DISCOVERY
1112.63855
9.902160645


20
16478
NM_020530.3
GE54528
DISCOVERY
663.3167725
6.018188477


21
52539
AI133415.1
GE82473
DISCOVERY
17346.24609
158.6896667


22
41803
NM_178313.1
GE735470
DISCOVERY
89.23596191
0.820007324


23
41817
NM_016292.1
GE79992
DISCOVERY
10618.77539
98.42697144


24
18884
NM_015604.2
GE57715
DISCOVERY
339.244873
3.387084961


25
8922
NM_024077.2
GE57504
DISCOVERY
10964.4248
110.8526306


26
24969
AV752332.1
GE60370
DISCOVERY
2446.138672
25.71212769


27
8634
BC043004.2
GE566269
DISCOVERY
947.6875
10.19564819


28
38879
NM_182832.1
GE476899
DISCOVERY
2933.067871
31.93505859


29
49586
BG289120.1
GE58687
DISCOVERY
248.4822998
2.715911865


30
11651
AW850450.1
GE85351
DISCOVERY
1144.841431
12.83999634


31
53172
NM_022749.4
GE59644
DISCOVERY
577.4226074
6.560333252


32
49622
CB052158.1
GE59962
DISCOVERY
3421.524414
39.68115234


33
22868
NM_152285.1
GE79323
DISCOVERY
539.8291016
6.525421143


34
23827
BG221408.1
GE59882
DISCOVERY
2403.98877
29.7802124


35
18139
AA938869.1
GE890404
DISCOVERY
2660.284912
33.11999512


36
22789
BX104097.1
GE61439
DISCOVERY
731.80696
9.725494385


37
11236
NM_003937.1
GE58794
DISCOVERY
4738.49707
65.4675293


38
10204
AW197778.1
GE81039
DISCOVERY
696
9.714294434


39
49170
AA577911.1
GE61247
DISCOVERY
713.4000244
10.09091187


40
42474
AW137161.1
GE62833
DISCOVERY
1164.558472
17.03775024


41
52342
NM_006147.2
GE54992
DISCOVERY
1200.007446
18.71737671


42
42573
NM_022648.2
GE79383
DISCOVERY
953.4078369
15.75


43
24440
NM_182936.1
GE592149
DISCOVERY
14
0.234771729


45
24440
NM_182936.1
GE592149
DISCOVERY
14
0.234771729


46
24440
NM_182936.1
GE592149
DISCOVERY
14
0.234771729


47
46186
NM_032746.1
GE82057
DISCOVERY
1407.181763
25.55319214


48
39926
INCYTE UNIQUE
GE82589
DISCOVERY
17409.49609
317.7109375


49
47689
AK097380.1
GE61992
DISCOVERY
3803.877441
72.08435059


50
12812
CF135919.1
GE55506
DISCOVERY
1176.72522
24.53225708


51
15994
R25284.1
GE79364
DISCOVERY
11397.33301
239.4736938


52
52071
BM698907.1
GE86827
DISCOVERY
415.7030029
9.42855835


53
40727
BG573885.1
GE55241
DISCOVERY
1204.090942
28.16665649


54
11635
AI376607.1
GE80662
DISCOVERY
106.1363525
2.5


55
4403
N26032.1
GE56202
DISCOVERY
891.694458
21.96551514


56
14949
BX110547.1
GE81738
DISCOVERY
1020.042969
25.26760864


57
51004
BM670853.1
GE59115
DISCOVERY
345.9705811
8.725006104


58
17177
AI274757.1
GE54607
DISCOVERY
1033.54187
26.16049194


59
9938
NM_152288.1
GE87537
DISCOVERY
11.78378296
0.299987793


60
21487
NM_006861.4
GE58813
DISCOVERY
387.6266479
9.909088135


61
37834
BU608350.1
GE57222
DISCOVERY
545.7602539
14.10638428


62
42661
C04533.1
GE60353
DISCOVERY
1506.339355
39.14474487


63
42680
AW449970.1
GE80314
DISCOVERY
1616.487305
42.02856445










B. Suppressed Gene












Suppression





Ratio
Description







1. Cancer-



Suppressing





Gene



1
461.4268293
heat shock protein 75 (TRAP1)



2
164.1818449
heat shock 70 kDa protein 14 (HSPA14)



3
116.6205651
AHA1, activator of heat shock 90 kDa protein ATPase





homolog 1 (yeast) (AHSA1)



4
 86.08481528
tumor rejection antigen (gp96) 1 (TRA1)



5
 84.26837836
brain specific protein (CGI-38)



6
 81.86905991
serologically defined breast cancer antigen 84





(SDBCAG84), transcript variant 1



7
 60.59218471
breast cancer metastasis suppressor 1 (BRMS1)



8
 53.65747638
Ras suppressor protein 1 (RSU1), transcript variant 1



9
 43.65344164
leukemia cell normalized cDNA library cDNA clone





LEU1757_26_C2



2. Immune



System Gene



1
172.891112
natural killer cell transcript 4 (NK4)



2
153.615681
CDS antigen, beta polypeptide 1 (p37) (CD8B1),





transcript variant 5



3
150.9221252
HLA-B associated transcript 5 (BAT5)



4
143.4498283
intercellular adhesion molecule 2 (ICAM2)



5
140.7596399
major histocompatibility complex, class I, E (HLA-E)



6
136.6714768
adhesion molecule AMICA (AMICA)



7
135.8569567
lymphotoxin beta (TNF superfamily, member 3) (LTB),





transcript variant 2



8
134.660619
apoptosis-associated speck-like protein containing a





CARD (ASC), transcript variant 1



9
130.1662446
HLA-B associated transcript 1 (BAT1), transcript





variant 1



10
129.1629268
killer cell lectin-like receptor subfamily K, member





1 (KLRK1)



11
122.6166802
IL2-inducible T-cell kinase (ITK)



12
121.8268278
tumor necrosis factor receptor superfamily, member 7





(TNFRSF7)



13
121.2896762
immediate early response 2 (IER2)



14
119.2151015
interferon (alpha, beta and omega) receptor 2





(IFNAR2), transcript variant 1



15
116.8236465
interferon, gamma-inducible protein 16 (IFI16)



16
115.4844704
granzyme A (granzyme 1, cytotoxic T-lymphocyte-





associated serine esterase 3) (GZMA)



17
113.1640763
immune associated nucleotide 2 (hIAN2)



18
112.365497
mal, T-cell differentiation protein (MAL), transcript





variant d



19
112.3632094
NK inhibitory receptor precursor (NKIR)



20
110.2186771
tumor necrosis factor (ligand) superfamily, member 12





(TNFSF12), transcript variant 1, mRNA



21
109.3092351
major histocompatibility complex, class II, DR alpha





(HLA-DRA)



22
108.8233718
programmed cell death 2 (PDCD2), transcript variant 2



23
107.8848128
major histocompatibility complex, class I, F (HLA-F)



24
100.1583595
(clone 38-1) MHC class I mRNA fragment.



25
 98.90991981
interferon induced transmembrane protein 1 (9-27)





(IFITM1)



26
 95.13559911
CD69 antigen (p60, early T-cell activation antigen)





(CD69)



27
 92.9501962
perforin 1 (pore forming protein) (PRF1)



28
 91.84476247
interferon stimulated gene 20 kDa (ISG20)



29
 91.49129726
interleukin-1 receptor-associated kinase 4 (IRAK4)



30
 89.16213062
implantation-associated protein (DKFZp564K142)



31
 88.01726761
transforming growth factor, beta 1 (Camurati-





Engelmann disease) (TGFB1)



32
 86.22542976
intercellular adhesion molecule 3 (ICAM3)



33
 82.72708991
lymphocyte cytosolic protein 2 (SH2 domain containing





leukocyte protein of 76 kDa) (LCP2)



34
 80.72436613
major histocompatibility complex, class II, DM alpha





(HLA-DMA)



35
 80.32262392
major histocompatibility complex, class II, DQ alpha





1 (HLA-DQA1)



36
 75.24645339
CASP8 and FADD-like apoptosis regulator (CFLAR)



37
 72.37934777
natural killer cell group 7 sequence (NKG7), mRNA



38
 71.64699451
interferon-related developmental regulator 1 (IFRD1)



39
 70.69728028
interferon gamma receptor 2 (interferon gamma





transducer 1) (IFNGR2)



40
 68.35165764
programmed cell death 6 (PDCD6)



41
 64.11194608
apoptosis-related protein PNAS-1 (FLJ39616)



42
 60.53383092
T-cell activation protein (PGR1)



43
 59.63239308
THYMUS cDNA clone CS0CAP007YL06 5-PRIME



45
 59.63239308
THYMUS cDNA clone CS0CAP007YL06 5-PRIME



46
 59.63239308
THYMUS cDNA clone CS0CAP007YL06 5-PRIME



47
 55.06872703
cell death-regulatory protein GRIM19 (GRIM19)



48
 54.79665331
thymosin-like 6 (TMSL6)



49
 52.7698094
killer cell lectin-like receptor subfamily B, member





1 (KLRB1)



50
 47.96644744
ovarian carcinoma immunoreactive antigen (OCIA)



51
 47.5932568
beta-2-microglobulin (B2M)



52
 44.08977359
interferon (alpha, beta and omega) receptor 1 (IFNAR1)



53
 42.74880629
linker for activation of T cells (LAT)



54
 42.45454102
thymostn, beta 4, Y-linked (TMSB4Y)



55
 40.595199
programmed cell death 4 (neoplastic transformation





inhibitor) (PDCD4), transcript variant 2



56
 40.36958872
programmed cell death 10 (PDCD10), transcript





variant 1



57
 39.65276092
tumor necrosis factor (ligand) superfamily, member





10 (TNFSF10)



58
 39.50773832
tumor necrosis factor superfamily, member 5-induced





protein 1 (TNFSF5IP1)



59
 39.28087487
interleukin 17D (IL17D)



60
 39.11829653
programmed cell death 2 (PDCD2), transcript variant 1



61
 38.68888322
proteasome (prosome, macropain) 26S subunit, non-





ATPase, 1 (PSMD1)



62
 38.4812664
proteasome (prosome, macropain) subunit, beta type,





9 (large multifunctional protease 2) (PSMB9),





transcript variant 1



63
 38.46163498
proteasome (prosome, macropain) 26S subunit, ATPase,





4 (PSMC4), transcript variant 1










This experiment revealed the facts described below, and it was clarified that the artificially synthesized peptide also significantly influenced the expression of human cancer-related gene to induce cancer, as in the peptide derived from the original cell line.
A. Enhanced Gene
1. Oncogene
Expression of oncogenes of cranial neuroblastoma, lung cancer, parotid gland tumor, breast cancer, colon cancer, renal cancer, ovarian cancer, melanoma, T-cell leukemia, B-cell leukemia, and so on were enhanced in the range of 47 to 1.7 times the baseline expression thereof. Expression of carcinogenic genes such as RAS, RAB, v-myc, and mab were also enhanced in the range of 6.7 to 1.7 times the baseline expression thereof. Additionally, expression of cell growth factor such as a platelet-derived growth factor was enhanced.
2. Immune System Gene
Expression of TNF, which is an inflammatory factor, was not enhanced, but expression of most of important factors as an operating factor for the humoral immunity mechanism, which is the opposite side of anti-tumor immunity, was enhanced.
B. Suppressed Gene
1. Cancer-suppressing Gene
Every cancer-suppressing gene was significantly suppressed: Expression of heat shock protein relating to cancer antigen-induction was suppressed in the range of 1/461 to 1/116 of the baseline expression thereof, and expression of a tumor rejection antigen was suppressed in the range of 1/86 to 1/81 of the baseline expression thereof. Expression of a gene relating to inhibition of metastasis of breast cancer was suppressed to 1/60, expression of a gene relating to inhibition of leukemia was suppressed to 1/43, and expression of RAS suppressor protein gene was suppressed to 1/43.
2. Immune System Gene
The expression of genes relating to anti-tumor immunity was highly suppressed as follows:
suppression of a gene relating to construction of thymus tissues or production of a thymic hormone;
suppression of a function and a construction factor of T-cell which is important in inhibition of cancer;
suppression of a function and construction of NK-cell;
suppression of production of a factor for killer-cell-secretion;
suppression of an apoptosis factor relating to cellular suicide;
suppression of a factor relating to cell adhesion in cell apoptosis;
suppression of production of antineoplastic interferon; and
suppression of expression of a cell organelle, an enzyme system, and a major histocompatibility complex (MHC) relating to induction of a cancer antigen.
(b) The peptide obtained in Example 3 was added to a culture medium (10 to 100 μg peptide/1×107 monocytes) for human normal peripheral blood monocytes extracted from subjects shown in Table 5 below, and they were cocultured for 7 hr. Then, total mRNA was extracted from the monocytes, and the amount of the total mRNA was measured and compared to that of the total mRNA before the treatment with the peptide. Table 5 shows the results.







TABLE 5







Effect of the artificially synthesized peptide on


total mRNA amount of peripheral blood monocytes












pre
post





total
total



RNA
RNA
post/pre


NAME
(μg/ml)
(μg/ml)
(%)
reduction ratio (%)















H. S.
45.875
0.958
2
98
Healthy subject







(smoker)


M. S.
20.779
9.188
44.2
55.8
Healthy subject







(smoker)


E.. E.
69.622
7.735
11.1
88.9
Healthy subject







(side-stream







smoke)


S. G.
61.145
16.562
27
73
Healthy subject


H. T.
66.804
14.742
22
78
Healthy subject







(side-stream







smoke)


M. O.
58.076
19.551
33.6
66.4
Healthy subject







(side-stream







smoke)


M. A.
52.318
17.681
33.7
66.3
Healthy subject


K. K.
8.078
1.757
21.7
78.3
Cancer patient







received







chemotherapy







and radio-







therapy





* Samples are equivalent to 1 × 107 peripheral blood monocytes.


* Treatment time with the peptide: 7 hr


* The processes for collecting total RNA are the same as those in Example 4.






With referred to the results shown in Table 5, the total mRNA in H.S. and E.E. were significantly decreased. H.S. was an addicted smoker who has smoked for more than ten years. With respect to M.S., the reduction ratio of total mRNA was low, but the pre-total mRNA amount itself was also low, which has been caused by smoking over a long period of time. E.E. was not a smoker, but father of E.E. was an addicted smoker. Therefore, it was assumed that the. decrease in the total mRNA was caused by passive smoking over a long period of time. Both H.T. and M.O. were passive smokers, and the reactivity to the peptide was accelerated in proportion to degree of the passive smoking (H.T.>M.O.). In K.K., the reduction ratio was low and the pre-total mRNA amount was significantly small. This subject was a cancer patient who has already received an anticancer agent and irradiation at the maximum possible dose. Therefore, the general gene translation was significantly impaired; which caused the result entirely different from that in healthy subjects. The results shown in Table 5 show that the cancer diagnosis can be efficiently performed by analyzing the changing ratio of total mRNA amounts before and after the treatment with the peptide of the present invention.
Example 5
Detection of the Peptide of the Present Invention in Cancer Cells and Influence of the Peptide on Normal Cells
In order to examine influences at a gene level of the peptide on normal cells, RT-PCR was performed by adding the peptide artificially synthesized in Example 3 to the normal cells.
(Method and Result)


    (1) Normal cells were treated with the artificially synthesized peptide for 7 hr as follows:

















Peptide



No. of Cells
Amount


















Kidney mesangium cell: (ACBRI-1376;
1.25 × 106
12.5 μg


Applied Cell Biology Research


Institute)


Skin fibroblast cell: (Catlog2F0-C25;
 4.0 × 106
40.0 μg


Cell Systems)


Pancreatic epithelial cells: (ACBRI515;
1.05 × 107
105.0 μg 


Applied Cell Biology Research Institute)


Peripheral blood monocytes:


H.S.
1.25 × 107
12.5 μg


M.S.
1.20 × 107
12.0 μg


E.E.
1.40 × 107
14.0 μg









(Kidney mesangium cells, skin fibroblast cells, and pancreatic epithelial cells: 100 μg peptide/1.0×107 cells, peripheral blood monocytes: 10 μg peptide/1.0×107 cells)

Then, total RNA was extracted from the above-treated normal cells, untreated normal cells, and squamous-cell carcinoma cells by using an RNA extraction kit (QIAGEN). The numbers of the treated cells and the untreated cells were the same.















RNA



No. of Cells
Amount


















Kidney mesangium cell: (ACBRI-1376; Applied
1.25 × 106
30 μg


Cell Biology Research Institute)


Skin fibroblast cell: (Catlog2F0-C25; Cell
 4.0 × 106
30 μg


Systems)


Pancreatic epithelial cells: (ACBRI515;
1.05 × 107
30 μg


Applied Cell Biology Research Institute)


Peripheral blood monocytes:


H.S.
1.25 × 107
60 μg


M.S.
1.20 × 107
60 μg


E.E.
1.40 × 107
60 μg


Cervical carcinoma cells (HeLa):
1.60 × 107
60 μg









Reverse transcription was performed by using 30 μg of each extracted total RNA to generate cDNA pools.
PCR was conducted using each of the cDNA pools as templates.
The condition for the PCR was as follows:
PCR buffer: 6 μl
dNTP: 2 μl
Primer Fw (11 amino acids or 7 amino acids): 1 μl
Primer Rv (oligo dT): 1 μl
dH2O: 9.5 μl
taq: 0.5 μl
Sample (RT products): 10 μl
(Total: 30 μl)
PCR: 45 cycles
Initial denaturation: at 94° C. for 5 min
Denaturation: at 94° C. for 30 sec
Annealing: at 55° C. for 30 sec
Extension: at 72° C. for 1 min

    (2) Each of the PCR products was applied to electrophoresis and detected by using a fluorescence label.
    (a) FIGS. 2A and 2B show the results when the full-length peptide (11 amino acids) of the present invention was used as the primer in the PCR.


The results are as follows:

    (i) Lanes 2 and 3 in FIG. 2A show the results of original UTC-8 cell samples which were not treated with the peptide of the present invention. The original UTC-8 cell samples stably expressed the gene of the preset invention.


Lane 4 in FIG. 2A shows the result of a cervical carcinoma cell sample, wherein a smear was observed. This means that the sample includes various sizes of genes which are complementary to the gene encoding the peptide of the present invention. Namely, the expression pattern of the gene was significantly changed in the cervical carcinoma cell sample by the treatment with the peptide of the present invention. Therefore, samples showing such a reaction pattern are thought to have a tendency of canceration.

    (ii) On the basis of the results above, samples on each lane were examined. In some samples (Lanes 5 and 6 of FIG. 2A, lanes 4 and 5, lanes 6 and 7, and lanes 8 and 9 in FIG. 2B), the original stable gene pattern was changed to a pattern having a broadened smear by the treatment with the peptide of the present invention. Such samples were determined to have a tendency of canceration. Actually, the blood sample of the lanes 5 and 6 of FIG. 2A was obtained from a young man who had smoked for more than 10 years. Though the samples were normal cells, they had a high sensitivity to the peptide. It is thought that such a high sensitivity is caused by that these cells were, as in the peptide of the present invention, extracted from epithelial cells and were derived from a tissue in an early stage. Therefore, the gene expression is not fixedly stable, and the unstable gene expression is involved in the acuity sensitivity against a highly disturbing factor from the outside such as the peptide of the present invention.
    (iii) On the contrary, in the sample of lanes 2 and 3 of FIG. 2B, gene mutation was hardly induced even if the sample was treated with the peptide of the present invention. This sample is thought to have a characteristic to rarely get cancer. Actually, the sample was obtained from a healthy young man in twenties.
    (b) FIGS. 3A and 3B show the results when a DNA encoding an amino acid sequence (7 amino acids) at positions 1-7 of the peptide of the present invention was used as a primer in the PCR.


The results are as follows:

    (i) Smears were observed in an UTC-8 cell sample shown in lane 3 of FIG. 3A and in a cervical carcinoma cell sample shown in lane 4 of FIG. 3A, as in the results when the full sequence was examined in the above-mentioned (a).
    (ii) In the samples of lanes 2 and 3, lanes 4 and 5 of FIG. 3B, smears were not observed even if the samples were treated with the peptide of the present invention, and normal patterns were observed.
    (iii) In the samples of lanes 5 and 6, lanes 7 and 8 of FIG. 3A and lanes 6 and 7, lanes 8 and 9 of FIG. 3B, gene expression patterns were significantly changed before and after the treatment with the peptide of the present invention.


As shown above, it was clarified that a tendency of canceration in normal cells can be determined by comparing changes in gene patterns induced by treating the cells with the peptide of the present invention.
In particular, when the PCR was performed by using the 11-amino acid primer (full sequence), a difference in the band patterns was observed in the peripheral blood sample (H.S.) and the skin fibroblast sample, and a difference in a position having a high concentration was observed in the kidney mesangium sample and the pancreatic epithelial sample, though it was a smear. When the PCR was performed by using the 7-amino acid primer (partial sequence), the band pattern of the pancreatic epithelial sample treated with the peptide for 7 hr was similar to that of the UTC-8 cell sample used as a control, and a difference in the band patterns was observed in the pancreatic epithelial sample by the treatment with the peptide for 7 hr.
A possible explanation for causes of such significant effects is that the UTC-8 cell is a cancer cell of epithelium and the peptide of the present invention is derived from UTC-8 cell line.
Therefore, epithelial cells are the most suitable samples for the measurement of a tendency of canceration using the peptide of the present invention, but it was proved that non-epithelial cell samples including peripheral blood, which can be most easily obtained in clinical practice, were useful for the diagnosis of a gene change in the full sequence.
Example 6
Exhaustive Gene Analysis of 55000 Genes by Using a DNA Chip System (Codelink System)
Characteristics of the peptide of the present invention were clarified by conducting the analysis described below in order to examine effects of the peptide on gene expression of normal cells.
(1) Preparation of Target cRNA
Normal lymphocytes were treated with the peptide of the present invention as in Examples 4 and 5, and the gene expression was compared to that of untreated lymphocytes by using a DNA chip system (Codelink System). In this examination, cRNA derived from bacteria was used as a positive control. Total RNA was extracted by using the QIAGEN RNA extraction kit used in Example 5. The total RNA (5 to 10 μg) was subjected to reverse transcription using an oligo-dT primer having a T7 RNA polymerase promoter region as a reverse primer to generate a pool of cDNAs complementary to the mRNAs. The condition for the reverse transcription was the same as that in Example 5. After second-strand cDNA synthesis, the treatment with T7 RNA polymerase and biotinylated rNTPs was performed in vitro transcription (IVT) reaction at 37° C. for 14 hr to obtain biotin-labeled cRNA. The amount of the cRNA was measured with a spectrophotometer to confirm at least 10 μg of the cRNA.
(2) Hybridization to Codelink Slide
The cRNA (10 μg) obtained in the above (1) was fragmentized and mixed with hybridization buffer, and then thermally denatured. This mixture solution was poured into a Codelink slide on which 55000 genes were immobilized, and a hybridization reaction was performed at 37° C. for 18 hr using an INNOVA 4080 shaker.
(3) Staining and Detection
The hybridization chamber was removed and the plate was washed at 46° C. for 1 hr. The hybridization was detected with Cy5-streptavidin (1:500 dilution, at room temperature for 30 min). The plate was further washed at a room temperature for 5 min 5 to 6 times and then washed with 0.05% Tween 20. The plate was dried by using a centrifuge for titer plates.
(4) The plate was scanned with an Arra WORK scanner with a PMT value of 600 at a resolution of 10 μm.
(5) The result was analyzed by exclusive software for the Codelink analysis. The software used was 1. Batch Submission 2 and 2. Codelink Expression 2.
Cancer-inducing expression status of cancer-related genes was observed in this experiment too, as in the results shown in Tables 1, 2, and 3.
Example 7
Examination of Antibody Against the Peptide for Cytotoxic Activity on Human Cancer Cells
Monoclonal antibody (prepared by using BALB/c mouse) against the peptide of the present invention was examined for cytotoxic activity on various human cancer cell lines shown in FIG. 4: Each cancer cell line was seeded in a 96-well plate at 1×104 cells/well, and cultured in 5% CO2 for 4 days to confirm that the cancer cells proliferated and adhered to the bottom surface of the plate so as to cover more than 80% of the surface area. The culture medium was removed by aspiration, and then 30 μl of the monoclonal antibody was added to each well under ice-cooling so that the surfaces of the cancer cells were brought into contact with the culture medium containing the monoclonal antibody. Then, the cancer cells were cultured under ice-cooling for 1 hr. As a control, each cancer cell line was also cultured in PBS buffer not containing the monoclonal antibody. Cytotoxic activity of the monoclonal antibody was examined by comparing the cancer cells treated with the monoclonal antibody with those of the control, and viability of the cell was determined by Trypan Blue staining. In the control, no substantial detachment of the cells from the bottom surface of the plate was observed; hence, cytotoxicity was not observed. On the contrary, in the cancer cells treated with the monoclonal antibody against the peptide of the present invention, detachment of the cells was observed in cancer cells shown in FIG. 4; hence, cytotoxic activity of the monoclonal antibody was observed. In addition, the detachment of the cancer cells was immediately induced by the addition of the monoclonal antibody, and the cancer cells were killed by the activity of the monoclonal antibody within only one hour. The photograph in the right of FIG. 4 shows human pancreatic cancer cells stained with FITC-labeled monoclonal antibody No. 1 of the present invention. These human pancreatic cancer cells died after this treatment.
Example 8
Immunosuppressive Agent Reducing Rejection in Transplantation
The experiment using the DNA chip proved that the peptide (SEQ ID NO: 1) had an ability to activate a cancer-related gene, and further suggested clinical usefulness of the cellular immunity-inhibition activity of the peptide. Then, the cellular immunity-inhibition activity of the peptide was examined from the viewpoint of activity for preventing rejection in skin transplantation.
The peptide of the present invention was administered to mice who have received skin transplantation according to the schedule shown in Table 6.









TABLE 6





Days from

Administration


Transplantation
Transplantation
of Peptide

















−3

Administration


−2

Administration


−1

Administration


0
Operation


1


2

Administration


3

Administration


4


5


6


7

Administration


8


9


10


11


12


13


14

Administration


15


16









    Subject: skin of the back of a C57Black/6N mouse was transplanted to the back of a BALB/c mouse
    Administration route: subcutaneously administered into the neck
    Administration amount: 50 μg peptide/0.1 ml/mouse
    Number of the subject: 6


The results show that, in the cross-transplantation of skin of the back of a C57Black/6N mouse to the back of a BALB/c mouse, the rejection in the skin transplantation was suppressed and skin graft survival was prolonged by intermittently administering the peptide at a very low amount of 50 μg peptide/0.1 ml/mouse through a subcutaneous route which is a most gentle administration (see the progress shown by the graph in FIG. 5). Namely, in the control, skin rejection occurred 7 days after the skin transplantation and the transplanted skin was rejected and was lost within 10 days after the transplantation in all 6 mice as shown in a photograph (control G1) of mice at bottom right in FIG. 5. On the contrary, in the mice administered with the peptide, skin rejection occurred 12 days after the transplantation; which was a 5 day delay. Furthermore, the transplanted skin of one mouse was survived for 14 days after the transplantation as shown in a photograph (treatment G1) at upper right in FIG. 5. Though the transplanted skin was lost in all mice on the 15th day from the date of the transplantation, a delay in the rejection in skin transplantation was observed.
Therefore, it was proved that the peptide can be used as an immunosuppressive agent by investigating administration dosage and route to increase efficiency thereof.

## Claims

1. An isolated or purified peptide having an ability to activate a cancer-related gene, wherein the peptide is obtained from cell membrane surfaces of human squamous-cell carcinoma cells and consists of the amino acid sequence represented by SEQ ID No: 1.

2. An isolated or purified peptide having an ability to activate a cancer-related gene, wherein the peptide has eleven amino acids and is obtained from cell line UTC-8 (FERM BP-08611) established from human squamous-cell carcinoma; and the peptide shows a detectable peak at a detection wavelength of 214 nm in samples collected during gel filtration of an extract of the cell line.

3. An immunosuppressive agent comprising the peptide according to claim 1 as an effective component.

4. A method for manufacturing a peptide according to claim 1, the method comprising the steps of:
immersing cells prepared by culturing human squamous-cell carcinoma cells in a culture medium into a citrate-phosphate buffer having a pH of 3.3 to 3.4 for extraction; and
fractionating the extract by gel filtration using a citrate-phosphate buffer having a pH of 6.8 to 7.0 for yielding a peptide having an ability to activate a cancer-related gene in human normal cells.

5. An immunosuppressive agent comprising the peptide according to claim 2 as an effective component.

6. A method for manufacturing a peptide according to claim 2, the method comprising the steps of:
immersing cells prepared by culturing human squamous-cell carcinoma cells in a culture medium into a citrate-phosphate buffer having a pH of 3.3 to 3.4 for extraction; and
fractionating the extract by gel filtration using a citrate-phosphate buffer having a pH of 6.8 to 7.0 for yielding a peptide having an ability to activate a cancer-related gene in human normal cells.