CN101273145A - Cancer related gene RASGEF1A - Google Patents

Abstract

The present invention provides methods for detecting and diagnosing cancer. According to an embodiment, the diagnostic method involves the determination of the expression level of the RASGEF1A gene which was discovered to discriminate cancer cells from normal cells. Furthermore, the present invention provides methods of screening for therapeutic agents useful in the treatment of cancer, methods for treating cancer, and methods for vaccinating a subject against cancer.

Description

Cancer related gene RASGEF 1 A

The application requires the rights and interests of U.S. Provisional Application that submit to, serial number 60/704,054 on July 28th, 2005, and its contents intact is collected herein by reference.

Technical field

The present invention relates to be used to detect with the method for diagnosing cancer and be used for the treatment of method with preventing cancer.

Background technology

Stones in intrahepatic bile duct cancer (ICC) is the primary hepatobiliary cancer of second common type.In Japan and western countries, especially in Britain, its sickness rate raise day by day (Shaib YH et al., J Hepatol 2004,40:472-7; Taylor-Robinson SD et al., Lancet 1997,350:1142-3; Kato I et al., Jpn JClin Oncol 1990,20:232-7).Furtherd investigate modal malignant tumor of liver-hepatocellular carcinoma, and obtained clinical progress by developing multiple therapeutic strategy.Yet, very few for the oncogenic process understanding that ICC involved, and ICC patient's prognosis is still bad.Aspect treatment, the surgical oncology surgical blanking almost is sole mode (the Okuda K et al. that overcomes this disease, J Gastroenterol Hepatol2002,17:1056-63), but the cancer cells of removing invasive ICC patient in late period fully is almost impossible.In addition, also unsatisfactory at the chemotherapy of ICC; For example, although have the part response rate of 20-30% based on the drug regimen of 5 FU 5 fluorouracil and gemcitabine, (Gores GJ, Hepatology 2003,37:961-9) not demonstrate survival benefit.Therefore, the new ICC therapeutic strategy of exploitation is extremely urgent.

The Ras family of GTP enzyme brings into play keying action at the signal integration from cell surface receptor to downstream effect device approach with transmitting.Ras albumen main GDP combined with non-activity in resting cell exists.Their interconvertible circulations between the GDP mating type of activated GTP mating type and non-activity (Hall A, Annu.Rev Cell Biol 1994,10:31-54).Their activity is subjected to regulate and control with the proteic interaction of adjusting that stimulates GDP/GTP exchange (GEF albumen) and GTP hydrolysis (GAP albumen).After the activation that is subjected to the extracellular stimulus thing, guanine nucleotide exchange factor (GEF) impels Ras to discharge GDP, and GTP can be combined with Ras.This GTP mating type Ras is formed with active conformation, and with the signal transmission with mediate the effect protein in downstream, such as Raf, phosphoinositide 3-kinase and RalGDS (Campbell SL et al., Oncogene 1998,17:1395-413; Quilliam LA et al., Bioessays1995,17:395-404).Though identified the many Ras GTP of family enzymes, it is limited to have illustrated active RasGEF number; For example, be Sos, GRF and GRP for Ras, be C3G, CalDAG1 and Epac for Rap1, and for Ral be the RalGDS family member (Quilliam LA et al., Bioessays 1995,17:395-404; Bos JL, EMBO J 1998,17:6776-82; De Rooij J et al., Nature 1998,396:474-7; Ebinu JO et al., Science 1998,280:1082-6; Gotoh T et al., J Biol Chem1997,272:18602-7; Kawasaki H et al., Proc Natl Acad Sci USA 1998,95:13278-83).

The genetic expression preface type that produces by the cDNA microarray analysis can provide than the significantly more details about the individual cancer characteristic of traditional histopathology method.The future of such information be clinical strategy that it might be used to improve the treatment neoplastic disease and developing new drug (Petricoin EF et al., Nat Genet2002,32Suppl:474-9).For this purpose, the inventor by the cDNA microarray analysis from expression preface type (Okabe H et al., Cancer Res 2001, the 61:2129-37 of the tumour of different tissues; Hasegawa S et al., Cancer Res 2002,62:7012-7; Kaneta Y et al., Jpn J CancerRes 2002,93:849-56; Kaneta, Y.et al., Int J Oncol 2003,23:681-91; Kitahara Oet al., Cancer Res 2001,61:3544-9; Lin Y et al., Oncogene 2002,21:4120-8; Nagayama S et al., Cancer Res 2002,62:5859-66; Okutsu J et al., Mol CancerTher 2002,1:1035-42; Kikuchi T et al., Oncogene 2003,22:2192-205).

Be designed for the evaluation that the research that discloses the cancer mechanism has promoted the molecule target of some antineoplastic agent.For example, the farnesyl transferase inhibitor (FTI) that growth signals pathway, its activation that initial exploitation is used for suppressing to relate to Ras depends on translation back farnesylation has demonstrated effective treatment Ras dependent tumors (Sun J et al. at animal model, Oncogene 1998,16 (11): 1467-73).Similarly, use the human clinical trial of antineoplastic agent and anti-HER 2 monoclonal antibody trastuzumab (trastuzumab) (for antagonism proto-oncogene acceptor HER2/neu) combination in the cancer patients, to realize clinical response and overall survival (the Molina MA et al. that makes moderate progress, Cancer Res 2001,61 (12): 4744-9).At last, the tyrosine kinase inhibitor STI-571 that has developed selective inactivation bcr-abl fusion rotein treats chronic granulocytic leukemia, and wherein the constitutively activate of bcr-abl Tyrosylprotein kinase is brought into play keying action in white corpuscle transforms.The medicament of these kinds is designed for the carcinogenic activity (O ' Dwyer ME ﹠amp that suppresses the specific gene product; Druker BJ, Curr Opin Oncol 2000,12 (6): 594-7).Therefore, the gene product that generally raises in cancer cells obviously can be served as the potential target that is used to develop new antineoplastic agent.

Further prove tumor associated antigen (TAA) institute's deutero-epitope peptide and the cracking tumour cell presented on CD8+ cytotoxic T lymphocyte (CTL) the identification MHC I quasi-molecule.The first routine TAA is a MAGE family, because this discovery uses immunological method to find many other TAA (Boon, Int J Cancer 1993,54:177-80; Boon ﹠amp; Van der Bruggen, J Exp Med 1996,183:725-9; Van der Bruggen et al., Science 1991,254:1643-7; Brichard et al., J ExpMed 1993,178:489-95; Kawakami et al., J Exp Med 1994,180:347-52).Some newfound TAA is carrying out the clinical development as the immunotherapy target now.The TAA that is found comprises MAGE (van der Bruggen et al. up to now, Science 1991,254:1643-7), gp100 (Kawakami et al., J Exp Med 1994,180:347-52), SART (Shichijo et al., J ExpMed 1998,187:277-88) and NY-ESO-1 (Chen et al., Proc Natl Acad Sci USA 1997,94:1914-8).On the other hand, proved that in tumour cell specificity crosses the gene product of expression and demonstrated as the target of inducing cell immunne response and discerned.This type of gene product comprise p53 (Umano etal., Brit J Cancer 2001,84:1052-7), HER2/neu (Tanaka et al., Brit J Cancer 2001,84:94-9), CEA (Nukaya et al., Int J Cancer 1999,80:92-7) etc.

Although in basis relevant and clinical study, obtained marked improvement (Rosenberg etal., Nature Med 1998,4:321-7 with TAA; Mukherji et al., Proc Natl Acad Sci USA 1995,92:8078-82; Hu et al., Cancer Res 1996 56:2479-83), can be used for treating gland cancer at present, comprises that the number of candidate TAA of colorectal carcinoma is still very limited.Abundant express and TAA that this expression is confined to cancer cells may be the ideal candidate of immunotherapy target in cancer cells.In addition, can induce the evaluation expectation of the new TAA of strong and specific anti-tumor immune response can promote clinical use (Boon et al., J Exp Med 1996, the 183:725-9 of peptide vaccination strategy in the broad variety cancer; Van derBruggen et al., Science 1991,254:1643-7; Brichard et al., J Exp Med 1993,178:489-95; Kawakami et al., J Exp Med 1994,180:347-52; Shichijo et al., J ExpMed 1998,187:277-88; Chen et al., Proc Natl Acad Sci USA 1997,94:1914-8; Harris, J Natl Cancer Inst 1996,88:1442-55; Butterfield et al., Cancer Res 1999,59:3134-42; Vissers et al., Cancer Res 1999,59:5554-9; Van der Burg et al., JImmunol1996,156:3308-14; Tanaka et al., Cancer Res 1997,57:4465-8; Fujieet al., Int J Cancer 1999,80:169-72; Kikuchi et al., Int J Cancer 1999,81:459-66; Oiso et al., Int J Cancer 1999,81:387-94).

According to repeatedly, reply peptide and produce the IFN-α of conspicuous level from the peripheral blood mononuclear cell (PBMC) that stimulates through peptide of some healthy donors, but ⁵¹Cr-discharge in the assay method seldom be confined to HLA-A24 or-mode of A0201 to tumour cell bring into play cytotoxicity (Kawano et al., CancerRes 2000,60:3550-8; Nishizaka et al., Cancer Res 2000,60:4830-7; Tamura etal., Jpn J Cancer Res 2001,92:762-7).Yet the two is HLA allelotrope (Date et al., Tissue Antigens 1996,47:93-101 common among Japanese and the Caucasian for HLA-A24 and HLA-A0201; Kondo et al., J Immunol 1995,155:4307-12; Kubo et al., J Immunol1994,152:3913-24; Imanishi et al., Proceeding of the eleventh InternationalHistocompatibility Workshop and Conference Oxford University Press, Oxford, 1992,1065; Williams et al., Tissue Antigen 1997,49:129).So, the cancer antigen peptide of being presented by these HLA may be useful especially for the cancer among treatment Japanese and the Caucasian.In addition, the peptide of known use high density is usually at the CTL of external evoked low-affinity, go up generation high-caliber particular peptide/MHC mixture at antigen presenting cell (APC), this mixture can effectively activate these CTL (Alexander-Miller et al., Proc Natl Acad Sci USA 1996,93:4102-7).

Summary of the invention

The present invention is based on the discovery of the specific expressed pattern of RASGEF1A gene in cancerous cells.

To the analysis of genome range (genome-wide) genetic expression preface type among the 25 routine ICC, identify one group of gene (Obama K et al., Hepatology 2005 Jun, 41 (6): 1339-48) that expression is generally raised by before.At this, focus on the gene RASGEF1A (RasGEF structural domain family, member 1A) in these genes.The inventor has detected the expression of RASGEF1A gene in bladder cancer and has raise (PCT/JP2006/302684), and the expression by inhibitation system in mammary cancer (WO 2005/28678).The present invention has further disclosed the RASGEF1A gene and has frequently raised in comprising stones in intrahepatic bile duct cancer (ICC), lung cancer, cancer of the stomach, colorectal carcinoma, prostate cancer, chronic myeloid leukemia, kidney, soft tissue sarcoma, carcinoma of the pancreas and seminomatous many people's tumours.In addition, find the protein enhancing RAS activity of this genes encoding.In addition, because siRNA (siRNA) causes the growth-inhibiting and/or the necrocytosis of ICC cell to the inhibition of RASGEF1A gene, this gene can be used as the new treatment target of dissimilar people's tumours.

At the RASGEF1A of this evaluation gene and transcribe with translation product and have diagnostic uses, can change its expression and/or activity and treat or relieving cancer symptoms as cancer markers and oncogene target.Similarly, can be used for treating or all cpds of preventing cancer by detecting the RASGEF1A expression of gene because of the change due to the compound, can identifying.

Therefore, the invention provides and a kind ofly be used for diagnosing or the method for definite experimenter's cancer proneness (predisposition), its biological sample that derives from the experimenter by mensuration is realized such as the RASGEF1A gene expression dose in the tissue sample.Gene expression dose rising compared with the normal control level shows that this experimenter suffers from cancer or risky generation cancer.Can use available from the normal cell of non-cancerous tissue and measure the normal control level.

In the present invention, preferred cancer is ICC.

In the context of the present invention, phrase " control level " refers to detected RASGEF1A gene expression dose in control sample, comprises normal control level and cancer control level.Control level can be to derive from single reference group's single expression pattern or derive from a plurality of expression patterns.For example, control level can be to come from the previous expression pattern database of subject cell." normal control level " refers to detected RASGEF1A gene expression dose in the individual crowd of the individual or known not cancer stricken of normal health.Normal individual is the individuality that does not have the cancer clinical symptom.On the other hand, " cancer control level " refers to suffer from the individuality of cancer or the RASGEF1A gene expression dose seen in the crowd.

The rising compared with the normal control level of detected RASGEF1A gene expression dose shows that this experimenter (sample source) suffers from cancer or risky generation cancer in the sample.

Perhaps, can compare in the interior one group of expression of gene level and the cancer control level of gene on the same group comprising the RASGEF1A gene in the sample.Similarly between sample expression level and the cancer control level show that this experimenter (sample source) suffers from cancer or risky generation cancer.

At this, if genetic expression is compared with control level and is raise or for example reduced by 10%, 25% or 50%, perhaps at least 0.1 times, at least 0.2 times, at least 1 times, at least 2 times, at least 5 times, at least 10 times or more times, think that then expression level has " change ".Can measure the RASGEF1A gene expression dose by the intensity for hybridization of genetic transcription thing in test example such as nucleic acid probe and the sample.

In the context of the present invention, the tissue sample that derives from the experimenter can be to obtain to suffer from any tissue that cancer or suspection suffer from the patient of cancer from the experimenter who accepts test is for example known.For example, tissue can comprise epithelial cell.More particularly, tissue can be carcinous epithelial cell.

The present invention further provides and be used to identify and suppress or strengthen the expression of RASGEF1A or the method for active compound, it is by contacting and measure the RASGEF1A gene expression dose with test-compound with the subject cell of expression RASGEF1A or the gene product activity realizes.Subject cell can be an epithelial cell, such as carcinous epithelial cell.Compare with the control level under not having the test-compound situation, gene expression dose or the active reduction of its gene product show that this test-compound can be used for alleviating cancer symptoms.

The present invention also provides a kind of test kit, its comprise at least a can with RASGEF1A genetic transcription or translation product bonded detection reagent.

Methods of treatment of the present invention comprises and is used for the treatment of or prevents method for cancer among the experimenter that it comprises the step of the experimenter being used the antisense composition.In the context of the present invention, the antisense composition reduces particular target gene (being the RASGEF1A gene) expression.For example, the antisense composition can contain and RASGEF1A gene order complementary Nucleotide.Perhaps, method of the present invention can comprise the step of the experimenter being used the siRNA composition.In the context of the present invention, the siRNA composition reduces RASGEF1A genetic expression.In another method, can carry out treatment for cancer or prevention among the experimenter by the experimenter being used the ribozyme composition.In the context of the present invention, nucleic acid specificity ribozyme composition reduces RASGEF1A genetic expression.In fact, the inventor has confirmed the restraining effect of siRNA to the RASGEF1A gene.For example, proved the inhibition of siRNA to the cell proliferation of cancer cells in the embodiment chapters and sections, it supports the fact of RASGEF1A gene as preferred cancer therapy target.

The present invention also comprises vaccine and method of vaccination.For example, a kind ofly be used for the treatment of or prevent method for cancer among the experimenter to comprise the experimenter is used the polypeptide that contains the RASGEF1A genes encoding or the segmental vaccine of immunologic competence of this polypeptide.In the context of the present invention, the immunologic competence fragment length is shorter than total length naturally exists protein, still can induce the polypeptide of the immunne response that is similar to this full length protein institute inductive immunne response.For example, the immunologic competence fragment length should have at least 8 amino-acid residues and can stimulate immunocyte such as T cell or B cell.Can measure the immunocyte stimulation by the processing generation of detection cell proliferation, cytokine (for example IL-2) or the generation of antibody.

An advantage of method described here is just can identify this disease before detecting tangible cancer clinical symptom.Other features and advantages of the present invention read following describe in detail and claim after the general obviously.

The accompanying drawing summary

Figure 1A has shown that D4223 (RASGEF1A) gene is organized at 13 kinds of ICC, the expression in non-carcinous bile duct cell mixture and the 5 kinds of healthy tissuess.Use one group of RASGEF1A gene-specific primer to carry out sxemiquantitative RT-PCR.Contrast as quantitative with beta-actin (ACTB) expression of gene.What Figure 1B had shown D4223 (RASGEF1A) gene organizes the Northern engram analysis more.

Fig. 2 has shown the Subcellular Localization of RASGEF1A.Fig. 2 A has shown the result of the fluorescence immunoassay cytochemical staining of the RASGEF1A that utilizes the band FLAG label that the NIH3T3 cell of expressing the RASGEF1A of band FLAG label through plasmid transfection carries out.With the protein of anti-FLAG mouse antibodies colored zone FLAG label, then manifest (upper right quarter) with the goat anti-mouse second antibody that is conjugated with FITC.Carry out counterstaining (upper left quarter) with DAPI pair cell nuclear.The pictorial display that merges is at lower left quarter.In Fig. 2 B,, analyzed the protein expression of band Flag label in the cell by the Western engram analysis.

Fig. 3 A has shown the colony forming assay method of the COS7 cell of the plasmid transfection through expressing RASGEF1A.With Jim Sa dyestuff (Giemsa) solution-dyed through pCAGGS-n3Fc-RASGEF1A, pCAGGS-n3Fc-RASGEF1A (-) or pCAGGS-n3Fc-Mock cells transfected.Fig. 3 B has shown the cell viability that uses the cell counting test kit to measure by the MTT assay method.Fig. 3 C has shown the expression of RASGEF1A gene in the cell of the plasmid transfection through expressing siRNA (si-B and si-E).With the plasmid (siEGFP) of expressing EGFP with simulate plasmid (stand-in) in contrast.Carry out sxemiquantitative RT-PCR and check the gene silencing effect of siRNA.Contrast as quantitative with the ACTB expression of gene.Fig. 3 D has shown the growth inhibitory effect that is designed for the siRNA that suppresses RASGEF1A.Use the cell counting test kit to measure the viability of SSP25 cell response siRNA.

Fig. 4 A has shown the dissociate result of assay method of K-Ras (upper left quarter), Ha-Ras (upper right quarter) and N-RAS (bottom) Nucleotide.Handle with reorganization GST (hollow square frame), SOS (being with hatched square frame) or RASGEF1A (filled box) back interactional with Ras [ ³H]-GDP with do not carry out the relative quantity that such result that processing obtained (shadowed square frame) compares.It is active that Fig. 4 B has shown that the RAS of RASGEF1A activates.Pass through GST-RafRBD " drop-down " (pull-down) assay method analyzed the extract of cell through pCAGGS-n3Fc-RASGEF1A or simulation plasmid transfection.Use anti-FLAG antibody to check the expression (left part) of RASGEF1A in cell by immunoblotting assay.With the GTP mating type Ras of reorganization Raf-1 albumen precipitation activated form, and use anti-general Ras antibody to carry out analyzing (right part) by the Western engram analysis.

Fig. 5 A has shown the result who uses the wound healing assay method that the COS7 cell through RASGEF1A or analog carrier transfection carries out.Scratching back 12 and 24 hours, and catching the presentation graphics of migrating cell.Fig. 5 B has shown that the Matrigel that uses same cell to carry out invades the result of assay method.Measured the average cell number that passes the Matrigel migration by the counting in 5 different visuals field.Obtained similar result by revision test.

Detailed Description Of The Invention

Unless expressly stated otherwise,, term " one and/kind ", " being somebody's turn to do " and " described " refer to " extremely herein Few one/kind ".

" separate " with the term of material (such as polypeptide, antibody, polynucleotides etc.) coupling and " pure Change " represent that this material is substantially free of at least a other materials that are included in the natural origin. Cause This, antibody separation or purifying refers to be substantially free of cellular material such as carbohydrate, lipid or its He is from the antibody of the contaminative protein of the cell or tissue in this protein (antibody) source, maybe when changing Learn the antibody that is substantially free of precursor or other chemicals when synthesizing. Term " is substantially free of thin Born of the same parents' material " comprise that wherein polypeptide is separated with cellular component separated from it or the cell that restructuring produces Described polypeptide preparation thing. Therefore, the polypeptide that is substantially free of cellular material comprise have be less than about 30%, 20%, the heterologous protein of 10% or 5% (by dry weight basis) (being also referred to as " contaminative protein " at this) The polypeptide preparation thing. When the polypeptide restructuring produced, it also preferably was substantially free of culture medium, comprises having The polypeptide preparation thing that is less than the long-pending culture medium of about 20%, 10% or 5% protein preparation object. When polypeptide leads to When crossing the chemical synthesis generation, it preferably is substantially free of precursor or other chemicals, comprises having Be less than about 30%, 20%, 10%, 5% (pressing dry weight basis) with the synthetic relevant chemistry of this protein before The polypeptide preparation thing of body or other chemicals. Can be for example by the dodecyl sulphur at the protein preparation thing Single band after the coomassie brilliant blue staining of acid sodium (SDS)-polyacrylamide gel electrophoresis and gel etc. Appearance show that the specified protein prepared product contains the polypeptide of separative or purifying. Preferred real at one Execute in the scheme, antibody of the present invention be separate or purifying.

" separation " or " purifying " nucleic acid molecules is such as the cDNA molecule, when passing through recombinant technique Can be substantially free of other cellular materials or culture medium during generation, or when chemical synthesis, can be substantially free of Precursor or other chemicals. In a preferred embodiment, the nuclear of code book invention antibody Acid molecule be separate or purifying.

Term " polypeptide ", " peptide " and " protein " are at this commutative amino acid residue polymer that is used in reference to. These terms are applicable to that wherein one or more amino acid residues are that residue or the non-natural of modifying exists Residue, such as the amino acid polymer of corresponding naturally occurring amino acid whose artificial chemistry analogies, with And naturally occurring amino acid polymer.

Term " amino acid " refers to naturally occurring and synthetic amino acid, and with naturally occurring ammonia The base similar amino acid analogue of acid function and amino acid analog thing. Naturally occurring amino acid is by heredity Those of cipher coding, and in cell adorned those (for example hydroxyproline, γ-carboxylics after the translation Base glutamate and O-phosphoserine). Phrase " amino acid analogue " refer to except the R base with modification or Outside the skeleton of modifying, (it is former to connect hydrogen to have the basic chemical structure identical with naturally occurring amino acid The alpha-carbon atom of son, carboxyl, amino and R base) compound (for example homoserine, nor-leucine, first Methyllanthionine, sulfoxide, methionine methyl sulfonium). Phrase " amino acid analog thing " refers to have and general ammonia The different structure of base acid but chemical compound with similar function.

At this, amino acid can with they generally know, the IUPAC-IUB biochemical nomenclature commission Trigram symbol or the one-letter symbol recommended represent.

Unless clearly statement is arranged in addition, term " polynucleotides ", " nucleotides ", " nucleic acid " and " nucleic acid branch The son " commutative use, and with amino acids seemingly, represent with generally accepted single-letter code. Class Be similar to amino acid, they contain nucleic acid polymers naturally occurring and that non-natural exists.

The present invention's part is based on sending out that RASGEF1A gene expression in kinds cancer patient's the cell raises Existing. The nucleotides sequence of people RASGEF1A gene is shown in SEQ ID NO:1, and also can obtain certainly GenBank registration number AK095136. At this, the RASGEF1A gene contain people RASGEF1A gene with And the RASGEF1A genes of other animals, described other animals comprise non-human primate, mouse, Rat, dog, cat, Ma Heniu, but be not limited thereto, and comprise the equipotential mutant and in other animals, send out The existing gene corresponding to this RASGEF1A gene.

The amino acid sequence of people RASGEF1A gene code is shown in SEQ ID NO:2, and also can obtain certainly GenBank registration number BAC04491. In the present invention, the polypeptide of RASGEF1A gene code is called " RASGEF1A " is sometimes referred to as " RASGEF1A polypeptide " or " RASGEF1A albumen ".

According to an aspect of the present invention, function equivalent is also included within the RASGEF1A polypeptide. At this, " function equivalent " of protein is the polypeptide with the BA that is equivalent to this protein. Change sentence Talk about, the polypeptide of any reservation RASGEF1A protein biology ability all can be used as this in the present invention The function equivalent of sample. Such function equivalent comprises that wherein one or more amino acid are replaced, lacks Lose, add or be inserted into the natural those polypeptides that exists in the amino acid sequence of RASGEF1A albumen. Or The person, described polypeptide can be to comprise sequence with the respective egg white matter to have at least about 80% homology and (also claim Be sequence homogeneity) the polypeptide of amino acid sequence. In other embodiments, described polypeptide can be The polynucleotides institute of hybridizing takes place in the natural nucleotide sequence that exists with the RASGEF1A gene under the stringent condition Coding.

Phrase " strict (hybridization) condition " refers to common in the complex mixture of nucleic acid, the nucleic acid molecules meeting With the hybridization of its target sequence, but can't detect condition with the hybridization of other sequences. Stringent condition is sequence Dependent and be different in varying environment. Longer sequence can be in higher temperature generation specificity Hybridization. Tijssen is seen in comprehensive guidance about nucleic acid hybridization, Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, " Overview of principles Of hybridization and the strategy of nucleic acid assays " (1993). Generally speaking, on rule Under fixed ionic strength, the pH, for specific sequence, stringent condition is chosen as and is lower than pyrolysis chain temperature (Tm) About 5-10 ℃. Tm is (under ionic strength, pH and the nucleic acid concentration of regulation) 50% and target complementation Probe and target sequence hybridization be in poised state (when target sequence exists when excessive, under Tm, 50% Probe occupy be in poised state) time temperature. Also can be by adding the destabilizing agent such as formamide Reach stringent condition. For selective or specific hybrid, positive signal should be at least 2 times of background, Be preferably 10 times of background hybridization. Exemplary stringent hybridization condition can be as follows: 50% formamide, 5x SSC and 1%SDS, at 42 ℃ of incubations, or 5x SSC, 1%SDS, at 65 ℃ of incubations, at 50 ℃ In 0.2x SSC and 0.1%SDS, wash.

Generally speaking, people know does not affect this egg to one or more amino acid whose modifications in the protein The function of white matter. Those skilled in the art will recognize that change single amino acids or fraction are amino acid whose right Indivedual interpolations of amino acid sequence, disappearance, insertion or to substitute be a kind of " the conservative modification ", it is to albumen The change of matter produces the protein with similar functions. Provide function class like the amino acid whose conservative form that substitutes Be well known in the art. For example, following eight groups each self-containedly for each other, substitute for conservative Amino acid:

1) alanine (A), glycine (G);

2) aspartic acid (D), glutamic acid (E);

3) asparagine (N), glutamine (Q);

4) arginine (R), Methionin (K);

5) Isoleucine (I), leucine (L), methionine(Met) (M), Xie Ansuan (V);

6) phenylalanine (F), tyrosine (Y), tryptophane (W);

7) Serine (S), Threonine (T); With

8) halfcystine (C), methionine(Met) (M) (referring to for example Creighton, Proteins 1984).

Conservative modified polypeptides like this is included in the RASGEF1A albumen of the present invention.Yet the present invention is not limited to this, and RASGEF1A albumen also can comprise non-conservative modification, as long as they keep the proteic at least a biologic activity of RASGEF1A.In addition, not getting rid of homologue between polymorphism variant, kind, reach through the protein of modifying is those coded protein of these proteinic allelotrope.

In addition, RASGEF1A gene of the present invention is contained the polynucleotide of the above-mentioned RASGEF1A protein function Equivalent of encoding.

Except as otherwise noted, employed all technical terms of this paper are identical with the general implication of understanding of one of ordinary skill in the art of the present invention with scientific terminology.Having under the situation of conflict, be as the criterion with this specification sheets (comprising definition).

I. diagnosing cancer:

I-1. the tendentious method that is used for diagnosing cancer or generation cancer

Find that RASGEF1A expression of gene specificity in the cancer patients raises.Therefore, in this genes identified and transcribe with translation product and have diagnosis effectiveness, and can come diagnosing cancer by the RASGEF1A expression of gene of measuring in the cell sample as the cancer sign.Specifically, the invention provides a kind of tendentious method that is used for diagnosing experimenter's cancer or cancer takes place by RASGEF1A expression of gene level among the mensuration experimenter.

Can include but not limited to ICC, bladder cancer, lung cancer, cancer of the stomach, colorectal carcinoma, prostate cancer, chronic myeloid leukemia, kidney, soft tissue sarcoma, carcinoma of the pancreas and spermocytoma by the cancer of the inventive method diagnosis.Method of the present invention is specially adapted to diagnose ICC.

According to a further aspect in the invention, be used to diagnose the proneness that at least a any above-mentioned cancer takes place.

In the context of the present invention, term " diagnosis " intention contains prediction and probability analysis.Method intention of the present invention is used for the clinical decision about treatment mode, comprises for treatment for cancer intervention, Case definition such as staging, reaches disease surveillance and supervision.

Treat to be preferably Mammals by the experimenter of the inventive method diagnosis.Exemplary Mammals includes but not limited to for example people, non-human primate, mouse, rat, dog, cat, Ma Heniu.

Preferably gathering biological sample from experimenter to be diagnosed diagnoses.Any biologic material all can be used as the biological sample of mensuration, as long as its purpose that comprises the RASGEF1A gene is transcribed or translation product.Biological sample includes but not limited to bodily tissue and liquid, such as blood, sputum and urine.Biological sample preferably contains and comprises epithelial cell colony, more preferably comprises carcinous epithelial cell or comes from the epithelial cell colony of suspecting for carcinous tissue.In addition, in case of necessity, but described cell purifying is used as biological sample then from the bodily tissue and the liquid that are obtained.

According to the present invention, in deriving from experimenter's biological sample, measure RASGEF1A expression of gene level.Can use means known in the art transcribing this expression level of mensuration on (nucleic acid) product level.For example, can pass through hybrid method (for example Northern blot hybridization) and use the quantitatively mRNA of RASGEF1A gene of probe.Can on chip or array, carry out such detection.The preferred expression level that uses array to detect the several genes (for example multiple cancer specific gene) that comprises RASGEF1A gene of the present invention.Those skilled in the art can utilize the sequence information of RASGEF1A gene to come (SEQ ID NO:1; GenBank registration number AK095136) the such probe of preparation.For example, the cDNA of RASGEF1A gene can be used as probe.In case of necessity, available suitable marker such as dyestuff and isotopic labeling probe, and can detect the expression of gene level by the intensity of hybridization marker.

In addition, can be by using the quantitative RASGEF1A gene transcription of primer product based on the detection method (for example RT-PCR) of amplification.Also can prepare such primer based on the sequence information of available this gene.For example, employed primer (SEQ ID NO:5 and 6 among the embodiment; Perhaps 7 and 8) can be used for the detection undertaken by RT-PCR, but the present invention is not limited to this.

Specifically, probe or the primer that is used for the inventive method hybridized with the mRNA of RASGEF1A gene under stringent condition, moderate stringent condition or low stringency condition.As used herein, phrase " strict (hybridization) condition " refers to that the condition of hybridization can not take place with other sequences with its target sequence for probe or primer.Stringent condition be sequence dependent and can be different under varying environment.Compare with shorter sequence, under comparatively high temps, can observe the specific hybrid of longer sequence.Generally speaking, under the ionic strength and pH of regulation, for specific sequence, the temperature of stringent condition is chosen as and is lower than about 5 ℃ of pyrolysis chain temperature (Tm).Tm is (under ionic strength, pH and the nucleic acid concentration of regulation) 50% and the probe of target complement sequence and target sequence are hybridized temperature when being in equilibrium state.Because target sequence exists with excessive usually, therefore under Tm, 50% probe occupies and is in equilibrium state.Usually, stringent condition can be wherein at pH 7.0-8.3, salt concn is lower than about 1.0M sodium ion, be generally about 0.01-1.0M sodium ion (or other salts), and be at least about 30 ℃ and be at least about those conditions of 60 ℃ for long probe or primer temperature for short probe or primer (for example 10-50 Nucleotide) temperature.Also can reach stringent condition by the destabilizing agent that adds such as methane amide.

Perhaps, for diagnosis of the present invention, can detect translation product.For example, can measure the proteic quantity of RASGEF1A.A kind of method that is used to measure as the protein quantity of translation product comprises the immunoassay of using this proteinic antibody of specific recognition.This antibody can be monoclonal or polyclonal.In addition, any fragment of this antibody or modified outcome (for example chimeric antibody, scFv, Fab, F (ab ') ₂, Fv etc.) all can be used for detecting, as long as such fragment keeps the proteic binding ability of RASGEF1A.The method that preparation is used for these type antibody of protein detection is well-known in the art, and any method all can be used among the present invention to prepare above-mentioned antibody and Equivalent thereof.

Detect the method for this gene expression dose as another kind based on RASGEF1A gene translation product, use and observe staining power by immunohistochemical analysis at the proteic antibody of RASGEF1A.That is, observe strong dyeing and show that this proteinic amount has increased, and the high expression level of while RASGEF1A gene.

In addition, can detect it based on the biologic activity of translation product.At this specifically, RASGEF1A albumen is proved to be has Ras Nucleotide dissociate activity, RAS enhanced activity, and relevant with the migration of cancer cells.Therefore, the proteic above-mentioned activity of RASGEF1A can be used as and has the proteic index of RASGEF1A in the biological sample.For example, can follow the described method of embodiment and detect above-mentioned activity.

In addition, outside RASGEF1A expression of gene level, also can measure for example known accuracy that the expression of gene level of differential expression is diagnosed with raising in ICC of other cancer related genes.

If raise or for example reduce by 10%, 25% or 50% by the control level of corresponding cancer marker gene; Or be increased to more than 1.1 times, more than 1.5 times, more than 2.0 times, more than 5.0 times, more than 10.0 times or more; Or be reduced at least 0.5 times, at least 0.2 times, at least 0.1 times or still less, can think that the expression level that comprises the cancer marker gene of RASGEF1A gene in the biological sample raises or reduces.

Collect and the sample of storage from the known experimenter of morbid state (carcinous or non-carcinous) before utilizing, use these biological subject product of imitating to measure control level simultaneously.Perhaps, can determine control level based on the result who is obtained from RASGEF1A gene expression dose in the known experimenter's of morbid state the sample who measures before analyzing by statistical method.In addition, control level can be from the database of the expression pattern of suffered examination cell before.In addition, according to an aspect of the present invention, RASGEF1A expression of gene level can compare with a plurality of control level in the biological sample, and described control level is measured from a plurality of with reference to sample.The preferred use measured from such control level with reference to sample, and this derives from the similar types of organization of types of organization of the biological sample of originating to the patient with reference to sample.In addition, preferably use the standard value of RASGEF1A expression of gene level among the known crowd of morbid state.Can obtain this standard value by any means known in the art.For example the scope of mean value ± 2S.D. or mean value ± 3S.D. can be used as standard value.

In the context of the present invention, the control level of measuring from known non-carcinous biological sample is called " normal control level ".On the other hand, if control level is measured from carcinous biological sample, then be called " carcinous control level ".

If RASGEF1A expression of gene level raises compared with the normal control level or is similar with carcinous control level, then diagnosable experimenter suffers from cancer or risky generation cancer, in addition, under the situation that the expression level of a plurality of cancer related genes compares, the similar experimenter of showing of genetic expression pattern suffers from cancer or risky generation cancer between sample and the carcinous reference.

Test organisms imitate between expression level and the control level of product difference can with respect to contrast nucleic acid for example housekeeping gene (its expression level is known not different because of the carcinous or non-carcinous state of cell) carry out stdn.Exemplary crt gene includes but not limited to beta-actin, Glycerose 3-phosphate dehydrogenase and ribosomal protein P1.

I-2. assessment of cancer is treated validity

The RASGEF1A gene of differential expression also can use for cancer treatment procedure that will be monitored between normal and cancerous cells, and the above-mentioned method that is used for diagnosing cancer also can be used for assessing the validity of treatment to cancer.Specifically, can derive from the experimenter's who stands to treat the cell RASGEF1A expression of gene level by mensuration and assess the validity of this treatment cancer.If desired, before the treatment, during and/or afterwards different time points obtain population of test from the experimenter.For example, can follow under the title above ' I-1. is used for diagnosing cancer or the tendentious method of cancer takes place ' described method and measure RASGEF1A expression of gene level.In the context of the present invention, the control level that preferably compares with detected expression level is measured the RASGEF1A genetic expression in the cell that is not exposed to described treatment.

If RASGEF1A expression of gene level is compared with the control level of measuring from normal cell or do not contain the cell mass of cancer cells, expression level is similar represents that then described treatment is effectively, and expression level difference represents that then the clinical effectiveness of this treatment or prognosis are more undesirable.On the other hand, if compare at measuring from cancer cells or the control level that contains the cell mass of cancer cells, expression level difference is then represented effective treatment, and expression level is similar represents that then clinical effectiveness or prognosis are more undesirable.

In addition, the RASGEF1A expression of gene level before and after the method according to this invention can relatively be treated is to assess the validity of this treatment.Specifically, obtain detected expression level in same experimenter's biological sample (i.e. level before the treatment) before relatively deriving from detected expression level in experimenter's the biological sample (i.e. treatment back level) and treatment beginning after the treatment.The reduction that level is compared before treatment back level and the treatment represents that described treatment is effectively, the rising that level is compared before treatment back level and the treatment or similarly represent that then clinical effectiveness or prognosis are more undesirable.

As used herein, term " effectively " expression treatment causes that the expression of gene that pathologic raises in the subject reduces, the expression of gene of pathologic downward modulation raises or size, morbidity (prevalence) or the metastatic potential of cancer reduce.When using described treatment, " effectively " refers to treat the formation that can postpone or prevent tumour, perhaps can postpone, prevents or alleviate at least a clinical symptom of cancer when preventative.Can use the standard clinical scheme to carry out the assessment of tumour situation among the experimenter.

In addition, can combine to determine the validity of treatment with any known method that is used for diagnosing cancer.For example, can be tested and appraised symptom and carry out cancer diagnosis unusually, described symptom unusually for example loses weight, stomachache, backache, anorexia, feel sick, vomiting and general malaise, weakness and jaundice.

I-3. assessment of cancer experimenter's prognosis

The above-mentioned method that is used for diagnosing cancer also can be used for assessing the prognosis of experimenter's cancer.Therefore, the present invention also provides a kind of method that is used for assessment of cancer experimenter prognosis.For example, can follow under the title above ' I-1. is used for diagnosing cancer or the tendentious method of cancer takes place ' described method and measure RASGEF1A expression of gene level.For example, derive from RASGEF1A expression of gene level in patient's the biological sample of a series of disease stages and can be used as the control level that will compare with the expression of gene level that the experimenter is detected.By comparing RASGEF1A expression of gene level and control level among the experimenter, can assess experimenter's prognosis.Perhaps, by comparing the pattern of time dependent expression level among the experimenter, can assess experimenter's prognosis.

For example, the rising compared with the normal control level of RASGEF1A expression of gene level shows that prognosis is more undesirable among the experimenter.On the contrary, compared with the normal control level similar of expression level shows that experimenter's prognosis is better.

II. test kit:

The present invention also provides the reagent that is used to detect cancer, can detect the reagent of RASGEF1A gene transcription or translation product, specifically, and the nucleic acid of combination of energy specificity or evaluation RASGEF1A gene transcript.For example, can specificity in conjunction with or the nucleic acid of identifying the RASGEF1A gene transcript comprise such as the oligonucleotide (for example probe and primer) that has with a part of complementary sequence of RASGEF1A gene transcript.Perhaps, but the antibody illustration as being used to detect the reagent of this gene translation product.Above described probe, primer and antibody can be used as the suitable example of mentioned reagent under the title ' I-1. is used for diagnosing cancer or the tendentious method of cancer takes place '.

In addition, can detect it based on the biologic activity of translation product.As shown in embodiment, RASGEF1A albumen is proved to be has Ras Nucleotide dissociate activity, RAS enhanced activity and relevant with the migration of cancer cells.Therefore, according to the present invention, for Ras Nucleotide dissociates assay method, the required material of migration that detects RAS activity or cancer cells also can be used as reagent and is used to detect RASGEF1A expression of gene level.

These reagent can be used for the diagnosis of above-mentioned cancer.The mensuration form of using these reagent can be to be all Northern hybridization well-known in the art or sandwich ELISA.

Detection reagent can test kit form pack together.For example, detection reagent can be packaged in the container separately.In addition, detection reagent can detect necessary other reagent with this and packs.For example, test kit can comprise as the nucleic acid of detection reagent or antibody (can combine with solid substrate, or separate packing with making it with matrix bonded reagent), contrast agents (male and/or feminine gender) and/or detectable.Also can comprise the specification sheets (for example printed instructions, audiotape, VCR, CD-ROM etc.) that is used to implement described mensuration in the test kit.

As an aspect of of the present present invention, the reagent that is used to detect cancer can be fixed in solid substrate such as porous bar (porous strip), to form the site that at least one is used to detect cancer.The measurement zone of porous bar or detection zone can comprise a plurality of sites, and detection reagent (for example nucleic acid) is all contained in each site.Test strip also can contain the site for feminine gender and/or positive control use.Perhaps, control site can be arranged on the bar different with test strip.Choose wantonly, different detection site can contain the immobilization detection reagent (for example nucleic acid) of different amounts, and promptly the amount of first detection site is more, and the amount in site is less subsequently.Add biological subject and imitate behind the product, the number of loci that demonstrates detectable signal provides the quantitative target of RASGEF1A expression of gene level in the sample.Detection site can be arranged to any suitable detected shape, is generally the strip or the point-like of crossing over the test strip width.

III. screening method:

Utilize polypeptide or its fragment or this gene transcription regulatory region of RASGEF1A gene, this genes encoding, can screen the medicament of the biologic activity that changes this expression of gene or this coded by said gene polypeptide.Above-mentioned medicament can be used as the medicine of treatment or preventing cancer.Therefore, the invention provides that the screening of the polypeptide that utilizes RASGEF1A gene, this genes encoding or its fragment or this gene transcription regulatory region is used for the treatment of or the method for the medicament of preventing cancer.

By screening method of the present invention isolating medicament be a kind of active medicament of estimating to suppress RASGEF1A expression of gene or this gene translation product, therefore, be a kind ofly to be used for the treatment of or to prevent owing to for example material standed for of the disease of cell proliferation disorders such as cancer.Such medicament estimates can treat or prevent to be selected from ICC, bladder cancer, lung cancer, cancer of the stomach, colorectal carcinoma, prostate cancer, chronic myeloid leukemia, kidney, soft tissue sarcoma, carcinoma of the pancreas and seminomatous cancer.Promptly the such medicament that screens by the inventive method is considered to have clinical benefit and can further tests the ability that it stops growth of cancer cells in animal model or experimenter.

In the context of the present invention, treat that the medicament of identifying by screening method of the present invention can be any compound or the composition that comprises several compounds.In addition, screening method according to the present invention is exposed to cell or proteinic to be subjected to the reagent agent can be simplification compound or combination of compounds.When using the compound combination in the method, compound can order or contact simultaneously.

In screening method of the present invention, can use any reagent agent that is subjected to, the for example product of cell extract, cell culture supernatant liquid, organism of fermentation, marine organism extract, plant milk extract, protein purifying or rough, peptide, non-peptide compound, synthetic micromolecular compound (comprising the nucleic acid construct thing, for example sense-rna, siRNA, ribozyme etc.) and natural compounds.Of the present inventionly be subjected to the reagent agent can use also that any in the several different methods obtains in the combinatorial library method known in the art, comprise that (1) biology library, the parallel solid phase of (2) space addressable or liquid phase library, (3) need the synthetic library method, (4) " pearl one compound " library method of deconvolution and the synthetic library method that (5) use affinity chromatography to select.The biology library method of using affinity chromatography to select only limits to peptide library, and other four kinds of methods are applicable to peptide, and non-peptide oligomer or compound small molecules library (Lam, Anticancer Drug Des 1997,12:145-67).The method in synthetic molecules library be found in (De Witt et al., Proc Natl Acad Sci USA1993,90:6909-13 in this area for example; Erb et al., Proc Natl Acad Sci USA 1994,91:11422-6; Zuckermann et al., J Med Chem 1994,37:2678-85; Cho et al., Science 1993,261:1303-5; Carell et al., Angew Chem Int Ed Engl 1994,33:2059; Carell et al., Angew Chem Int Ed Engl 1994,33:2061; Gallop et al., J Med Chem 1994,37:1233-51).Library of compounds can exist in solution (referring to Houghten, Bio/Techniques 1992,13:412-21) or be present in pearl (Lam, Nature 1991,354:82-4), chip (Fodor, Nature1993,364:555-6), bacterium (U.S. Patent No. 5,223,409), spore (U.S. Patent No. 5,571,698; 5,403,484 and 5,223,409), plasmid (Cull et al., Proc Natl Acad Sci USA 1992,89:1865-9) or phage (Scott and Smith, Science 1990,249:386-90; Devlin, Science1990,249:404-6; Cwirla et al., Proc Natl Acad Sci USA 1990,87:6378-82; Felici, J Mol Biol 1991,222:301-10; U.S. Patent application 2002103360) on.

Wherein the part-structure by the compound that any screening method of the present invention screened through add, disappearance and/or replace change the compound that obtains and be included in the medicament that obtains by screening method of the present invention.

In addition, when the screening be subjected to the reagent agent to be protein the time, in order to obtain this protein DNA of encoding, can measure this proteinic complete amino acid sequence to release this nucleic acid sequences to proteins of coding, maybe can analyze the proteinic partial amino-acid series that obtained preparing few DNA as probe based on this sequence, and with this probe screening cDNA library to obtain this protein DNA of coding.The DNA that is obtained is used for preparation and is subjected to the reagent agent, and it is to be used for the treatment of or the material standed for of preventing cancer.

III-1. based on proteinic screening method

According to the present invention, proposition RASGEF1A expression of gene is vital for the growth and/or the survival of cancer cells.Therefore, think the growth and/or the survival of medicament anticancer of the function that suppresses this coded by said gene polypeptide, and can be used for treatment or preventing cancer.Therefore, the invention provides and utilize the RASGEF1A polypeptide to screen to be used for the treatment of or the method for the medicament of preventing cancer.

Outside the RASGEF1A polypeptide, the fragment of this polypeptide also can be used for screening of the present invention, as long as it keeps the natural at least a biologic activity that has the RASGEF1A polypeptide.

Described polypeptide or its fragment can further connect other materials, as long as this polypeptide and fragment keep its at least a biologic activity.Available material comprises: peptide, lipid, sugar and sugar chain, ethanoyl, natural and synthetic polymer etc.The modification that can carry out these types is to give other function of this polypeptide and fragment or to make it stable.

Be used for the polypeptide of the inventive method or fragment and can be used as and naturally exist protein to pass through conventional purification process to obtain, or obtain by chemosynthesis based on selected aminoacid sequence from nature.For example, can be used for the conventional method of peptide synthesis of synthetic comprises:

1)Peptide?Synthesis，Interscience，New?York，1966；

2)The?Proteins，Vol.2，Academic?Press，New?York，1976；

3) Peptide Synthesis (Japanese), Maruzen Co., 1975;

4) Basics and Experiment of Peptide Synthesis (Japanese), Maruzen Co., 1985;

5) Development of Pharmaceuticals (second volume) (Japanese), Vol.14 (peptide synthesis), Hirokawa, 1991;

6) WO99/67288; With

7)Barany?G.&?Merrifield?R.B.，Peptides?Vol.2，“Solid?Phase?Peptide?Synthesis”，Academic?Press，New?York，1980，100-118。

Perhaps, can adopt any known engineering method that is used to produce polypeptide to obtain this protein (Morrison J. for example, J Bacteriology 1977,132:349-51; Clark-Curtiss ﹠amp; Curtiss, Methods in Enzymology (editor Wu et al.) 1983,101:347-62).For example, but at first prepare the carrier that is fit to that comprises the polynucleotide of the target protein of encoding with expression-form (for example being positioned at the downstream of the adjusting sequence that comprises promotor), be transformed into proper host cell then, then cultivate host cell to produce protein.In particular, the gene by the RASGEF1A polypeptide of will encoding inserts carrier such as pSV2neo, pcDNAI, pcDNA3.1, pCAGGS or the pCD8 that is used to express alien gene, this gene of expression in host (for example animal) cell etc.Promotor can be used for expressing.Can use any promotor commonly used, comprise for example SV40 early promoter (Rigby in Williamson (volume), GeneticEngineering, vol.3.Academic Press, London, 1982,83-141), EF-α promotor (Kimet al., Gene 1990,91:217-23), the CAG promotor (Niwa et al., Gene 1991,108:193), RSV LTR promotor (Cullen, Methods in Enzymology 1987,152:684-704), SR α promotor (Takebe et al., Mol Cell Biol 1988,8:466), CMV immediate early promoter (Seed etal., Proc Natl Acad Sci USA 1987,84:3365-9), the SV40 late promoter (Gheysen et al., J Mol Appl Genet 1982,1:385-94), gland virus stage starting (Kaufman et al., MolCell Biol 1989,9:946), HSV TK promotor etc.Can carrier be imported host cell to express the RASGEF1A gene according to any method, electroporation (Chu et al. for example, Nucleic Acids Res 1987,15:1311-26), calcium phosphate method (Chen et al., Mol Cell Biol 1987,7:2745-52), DEAE dextran method (Lopata et al., Nucleic Acids Res 1984,12:5707-17; Sussman et al., Mol Cell Biol 1985,4:1642-3), lipofection (Derijard B, Cell 1994,7:1025-37; Lamb et al., Nature Genetics 1993,5:22-30; Rabindran et al., Science 1993,259:230-4) etc.

Also can adopt external translating system at external generation RASGEF1A albumen.

To can be polypeptide, the soluble protein of for example purifying or the fusion rotein that merges with other polypeptide with the RASGEF1A polypeptide that contacted by the reagent agent.

III-1-1. identify can with RASGEF1A polypeptide bonded medicament

Can may change this proteinic expression of gene of coding or this proteinic biologic activity with the medicament of protein bound.Therefore, as on the one hand, the invention provides that a kind of screening is used for the treatment of or the method for the medicament of preventing cancer, comprise step:

A) contacted with RASGEF1A polypeptide or its fragment by the reagent agent;

B) detect polypeptide and be subjected to combining between the reagent agent; And

C) select to be subjected to the reagent agent with the polypeptide bonded.

Can for example detect with combining of RASGEF1A polypeptide by the reagent agent by the immunoprecipitation of use at the antibody of this polypeptide.Therefore, in order to carry out above-mentioned detection, the RASGEF1A polypeptide or its fragment that are preferred for screening contain the antibody recognition site.The antibody that is used to screen can be the antibody of the antigenic region (for example epi-position) of identification RASGEF1A polypeptide of the present invention, and its preparation method is well-known in the art, and any method all can be used for preparing among the present invention above-mentioned antibody and Equivalent thereof.

Perhaps, RASGEF1A polypeptide or its fragment can be used as its N-or C-end comprise monoclonal antibody recognition site (epi-position) fusion rotein and obtain expressing, the specificity of described monoclonal antibody is revealed to be at the N-of this polypeptide or C-end.Can use commercially available epitope-antibody system (Experimental Medicine 1995,13:85-90).The carrier of the fusion rotein of beta-galactosidase enzymes, maltose binding protein, glutathione S-transferase, green fluorescent protein (GFP) etc. is commercial available by utilizing its multiple clone site to express for example to have, and can be used for the present invention.In addition, the also known fusion rotein that contains much smaller epi-position in this area, described epi-position can by detected with the immunoprecipitation of antibody at it (Experimental Medicine 1995,13:85-90).The above-mentioned epi-position of forming, can not change RASGEF1A polypeptide or its segmental characteristic by several to dozens of amino acid also can be used for the present invention.Example comprises polyhistidyl (His label), influenza virus aggregate (influenza aggregate) HA, people c-myc, FLAG, vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 albumen (T7 label), human herpes simplex vicus's glycoprotein (HSV label), E label (a kind of epi-position on the mono-clonal phage) etc.

As everyone knows, glutathione S-transferase (GST) also is the counterpart (counterpart) as the fusion rotein that will detect by immunoprecipitation.To merge when forming the protein of fusion rotein with RASGEF1A polypeptide or its fragment when GST is used as, availablely promptly detect this fusion rotein such as gsh (for example gsh-Sepharose 4B) at the antibody of GST or with GST specificity bonded material.

In immunoprecipitation, by adding antibody (identification RASGEF1A polypeptide or its fragment self, or the epi-position of mark on this polypeptide or the fragment) to the RASGEF1A polypeptide be subjected to form immunocomplex in the reaction mixture of reagent agent.If had and this polypeptide bonded ability by the reagent agent, so formed immunocomplex should be by the RASGEF1A polypeptide, formed by reagent agent and antibody.Otherwise if be subjected to the reagent agent to lack aforementioned capabilities, so formed immunocomplex only is made up of RASGEF1A polypeptide and antibody.Therefore, can be subjected to reagent agent and RASGEF1A polypeptide bonded ability by the size inspection of for example measuring formed immunocomplex.Any method that is used for the detection material size all can be used, and comprises chromatography, electrophoresis etc.For example, when mouse IgG antibody was used to detect, albumin A or Protein G sepharose can be used for quantitative formed immunocomplex.

Immunoprecipitation sees for example Harlow et al. for details, Antibodies, Cold Spring HarborLaboratory publications, New York, 1988,511-52.

In addition, be used to screen and combine with carrier with RASGEF1A polypeptide or its fragment of its bonded medicament.Can be used for comprising insoluble polysaccharide, such as agarose, Mierocrystalline cellulose and dextran in conjunction with the example of the carrier of polypeptide; And synthetic resins, such as polyacrylamide, polystyrene and silicon; Preferred commercially available pearl and the plate (for example porous plate, biologic sensor chip etc.) that uses by the above-mentioned materials preparation.When using pearl, it can be inserted pillar.Perhaps, also known in the art use magnetic bead can be easy to polypeptide and the medicament of separation and combination on pearl by magnetic.

Polypeptide can carry out according to conventional methods with combining of carrier, such as chemical bonding and physical adsorption.Perhaps, polypeptide can combine with carrier by the proteinic antibody of specific recognition.In addition, can also be by means of interactional molecule such as affinity the plain and combination of vitamin H carry out combining of polypeptide and carrier.

Utilize above-mentioned carrier mating type RASGEF1A polypeptide or its segmental screening to comprise and contacted with carrier mating type polypeptide by the reagent agent, this mixture of incubation, wash vehicle, and detection and/or measurement and carrier-bound medicament.Can in damping fluid, carry out this combination, such as but not limited to phosphate buffered saline buffer and Tris damping fluid, as long as described damping fluid does not suppress this combination.

For wherein being used as the screening method that is subjected to the reagent agent with above-mentioned carrier mating type RASGEF1A polypeptide or its fragment and composition (for example cell extract, cell lysate etc.), such method is commonly called affinity chromatography.For example, the RASGEF1A polypeptide can be fixed on the carrier of affinity column, will contain then and can put on this pillar with the reagent agent that is subjected to of this polypeptide bonded material.Be subjected in the reagent agent behind the sample, the washing pillar is then with suitable buffer solution elution and polypeptide bonded material.

In the present invention, utilize the biosensor of surface plasma resonance can be used as to detect or quantitatively in conjunction with the means of medicament.When using such biosensor, only use a spot of polypeptide, and need not mark (BIAcore for example, Pharmacia), can with the RASGEF1A polypeptide and to be subjected to the interaction Real Time Observation between the reagent agent be the surface plasma body resonant vibration signal.Therefore, utilization might be assessed polypeptide and be subjected to combining between the reagent agent such as the biosensor of BIAcore.

Be exposed to following molecule by the specified protein that will be fixed on the carrier, the method of screening and specified protein bonded molecule in the molecule in synthetic compound or crude substance storehouse or random phage peptide display libraries, and based on combinatorial chemistry technique (Wrighton et al., Science 1996,273:458-64; Verdine, Nature 1996, and the high-throughput screening method that 384:11-3) comes isolated protein and compound also is that those skilled in the art are well-known.These methods also can be used for the screening can with RASGEF1A albumen or its fragment bonded medicament (comprising agonist and antagonist).

When being subjected to the reagent agent to be protein, for example (Skolnik et al., Cell1991 65:83-90) can be used for method of the present invention to the West-Western engram analysis.Specifically, can express at least a and proteinic cell of RASGEF1A polypeptide bonded by expection by at first utilizing phage vector (for example ZAP), tissue, organ or culturing cell (for example SSP25) preparation cDNA library, on the LB-agarose, express protein by the vector encoded in cDNA library, expressed proteinaceous solid is fixed on the filter membrane, allow the RASGEF1A polypeptide and the above-mentioned filter membrane of process purifying and mark react, and detect according to the marker of RASGEF1A polypeptide and to express and the proteinic plaque of RASGEF1A polypeptide bonded, thereby obtain can with RASGEF1A polypeptide bonded protein.

In the method for the invention, mark substance such as radio isotope (for example ³H, ¹⁴C, ³²P, ³³P, ³⁵S, ¹²⁵I, ¹³¹I), enzyme (for example alkaline phosphatase, horseradish peroxidase, beta-galactosidase enzymes, beta-glucosidase enzyme), fluorescent substance (for example fluorescein isothiocyanate (FITC), rhodamine) and vitamin H/affinity element can be used for the mark of RASGEF1A polypeptide.When protein is used labelled with radioisotope, can detect or measure by liquid flashing.Perhaps, when protein was used enzyme labelling, the enzymatic change that can detect substrate by the substrate that adds enzyme detected or measures such as utilizing absorption spectrophotometry (absorptiometer) detection color to produce.In addition, be used as at fluorescent substance under the situation of marker, can utilize fluorophotometer to detect or measure institute's bonded protein.

In addition, can be by utilizing specificity in conjunction with the RASGEF1A polypeptide or merge to detect or measure and be bonded to proteinic RASGEF1A polypeptide to the antibody of the peptide of RASGEF1A polypeptide or polypeptide (for example GST).Use under the situation of antibody in the present invention's screening, this antibody preferably carries out mark with one of above-mentioned mark substance, and detects or measure based on this mark substance.Perhaps, can be used as first antibody at the antibody of RASGEF1A polypeptide, this antibody detects with the second antibody that is marked with mark substance.In addition, can utilize Protein G or albumin A post to detect or measure in the present invention screening and RASGEF1A polypeptide bonded antibody.

Perhaps, in another embodiment of screening method of the present invention, (" MATCHMAKER two-hybrid system ", " test kit is measured in Mammals MATCHMAKER double cross ", " MATCHMAKER single crosses system " are (Clontech) can to use the two-hybrid system that utilizes cell; " HybriZAP double cross carrier system (Stratagene) "; Reference Dalton et al., Cell 1992,68:597-612 and Fieldset al., Trends Genet 1994,10:286-92).In two-hybrid system,, and in yeast cell, express RASGEF1A polypeptide or its fragment and SRF land or the fusion of GAL4 land.Express at least a and the proteinic cell preparation cDNA of RASGEF1A polypeptide bonded library by expection, make this library when expressing with VP16 or the fusion of GAL4 transcription activating district.Then the cDNA library is imported above-mentioned yeast cell, and go out to derive from the cDNA in described library (when yeast cell, expressing can be with RASGEF1A polypeptide bonded protein the time from the clone and separate of test positive, both combinations have activated reporter gene, and positive colony can be detected).By above-mentioned isolating cDNA is imported intestinal bacteria and marking protein, can prepare by this cDNA encoded protein matter.

As reporter gene, outside the HIS3 gene, can use for example Ade2 gene, lacZ gene, CAT gene, luciferase genes etc.

Medicament by this screening and separating is the agonist of RASGEF1A polypeptide or the material standed for of antagonist.Term " agonist " thus refer to by combine the molecule that activates its function with polypeptide.In contrast, term " antagonist " refers to by combine the molecule that suppresses its function with polypeptide.In addition, by this screening as antagonist and isolating medicament is to suppress RASGEF1A polypeptide and the interior interactional material standed for of molecule (comprising nucleic acid (RNA and DNA) and protein) body.

III-1-2. identify medicament by the biologic activity that detects the RASGEF1A polypeptide

According to the present invention, it is vital that the RASGEF1A expression of gene demonstrates for the growth of cancer cells and/or survival.Therefore, be considered to can be as being used for the treatment of or the material standed for of preventing cancer for containment or the medicament that suppresses the biological function of RASGEF1A gene translation product.Therefore, the present invention also provides a kind of RASGEF1A of utilization polypeptide or its fragment to screen to be used for the treatment of or the method for the compound of preventing cancer, comprises the steps:

A) contacted with RASGEF1A polypeptide or its fragment by the reagent agent;

B) biologic activity of the polypeptide of detection step (a); And

Any polypeptide all can be used for screening, as long as it has a kind of biologic activity of RASGEF1A polypeptide, described biologic activity can be used as the index of screening method of the present invention.Because the RASGEF1A polypeptide has the activity that promotes cancer cell multiplication, the biologic activity that therefore can be used as the RASGEF1A polypeptide of screening index comprises the cell-proliferation activity of above-mentioned people RASGEF1A polypeptide.For example, but end user RASGEF1A polypeptide, also can use comprise its fragment, with its polypeptide of equal value on function.Can be by the endogenous or heterogenous expression aforementioned polypeptides of cell that is fit to.

When the biologic activity that is detected in the method for the present invention is cell proliferation, can be as described in the embodiment (referring to " material and method "), detect by the following method: RASGEF1A polypeptide or its segmental cell are expressed in preparation, be subjected to cultivate under the condition of reagent agent described cell in existence, and the mensuration cell proliferation rate, measure the cell cycle etc., and measure the wound healing activity, carry out Matrigel and invade assay method, and the measurement colony forms activity.

According to an aspect of the present invention, described screening further comprises step afterwards in above-mentioned steps (b):

C) select with do not have the condition that is subjected to the reagent agent under detected biologic activity compare suppress the polypeptide biologic activity be subjected to the reagent agent.

By this screening isolating medicament be the antagonist material standed for of RASGEF1A polypeptide, therefore, also be to suppress this polypeptide and the interior interactional material standed for of molecule (comprising nucleic acid (RNA and DNA) and protein) body.

As another aspect of the present invention, the biologic activity that can utilize the RASGEF1A polypeptide is screened medicament as the agonist material standed for of this polypeptide as index.According to aforesaid method, select the medicament of the biologic activity of raising RASGEF1A polypeptide.Specifically, this screening method further comprises step afterwards in above-mentioned steps (b):

C) select with do not have the condition that is subjected to the reagent agent under detected biologic activity compare improve the polypeptide biologic activity be subjected to the reagent agent.

At this, disclosed the activity of RASGEF1A polypeptide raising RAS.Therefore, the medicament of reduction RAS enhanced activity (being one of biologic activity of RASGEF1A polypeptide) can be used for treatment or preventing cancer.Therefore, the present invention further provides that a kind of screening is used for the treatment of or the method for the medicament of preventing cancer.An embodiment of this screening method comprises step:

(a) be subjected under the condition of reagent agent in existence, RASGEF1A polypeptide or its fragment are contacted with RAS;

(b) detect the RAS activity; And

(c) select with do not have the condition that is subjected to the reagent agent under detected activity compare and reduce that RAS is active to be subjected to the reagent agent.

Can come measure R AS activity according to means known in the art; For example, by GDP detect (referring to for example Hall BE et al., J Biol Chem 2001,276:27629-37) or RAS activate assay method.In the present invention, be subjected to the RAS of RASGEF1A polypeptide under the condition that whether the reagent agent exist to activate active in order to be evaluated at, for example, under the condition whether test-compound exists, will express cell lysate and GST-Ras fusion rotein, GDP and the GTP γ S incubation of the cell of RASGEF1A gene.Behind the incubation, the Western engram analysis of the anti-Ras antibody by using detection of active or 21kDa GTP-Ras comes detection of active or GTP-Ras.Can use gsh dish (glutathione disc) to carry out the drop-down assay method of activity or GTP-Ras.Commercially available RAS activates the mensuration test kit and also can be used for screening method of the present invention.For example, " StressXpress

The RAS activation kit " (Stressgen Bioreagents Corp.) or " EZ-DetectTM Ras activation kit " (PIERCE Biotechnology) comprise all components that is used for this mensuration, for example anti-RAS antibody, gsh dish, contains gst fusion protein, GDP and the GTP γ S of the Ras of Rafl in conjunction with territory (RBD).In addition, can be as detecting RAS activity (referring to " the 8.Ras Nucleotide dissociate assay method " and " 9.RAS activation analysis " of title " material and method ") as described in the embodiment.

III-2. based on the screening method of Nucleotide

III-2-1. use the screening method of RASGEF1A gene

As detailed above, by control RASGEF1A expression of gene level, can control the morbidity and the progress of cancer.Therefore, can be used for the treatment of or the medicament of preventing cancer by using RASGEF1A expression of gene level can identify as the screening of index.In the context of the present invention, such screening can comprise for example following steps:

A) will be subjected to reagent agent and the cells contacting of expressing the RASGEF1A gene;

B) detect RASGEF1A expression of gene level; And

C) select with do not have the condition that is subjected to the reagent agent under detected level compare reduce RASGEF1A expression of gene level be subjected to the reagent agent.

Can contact with being subjected to the reagent agent by the cell that will express the RASGEF1A gene, measure RASGEF1A expression of gene level then, thereby identify the active medicament that suppresses RASGEF1A expression of gene or its gene product.Certainly, the cell that also can use a group to express this gene replaces individual cells to carry out this evaluation.Under the condition that has this medicament, detect the expression level of comparing reduction with the expression level under the condition that does not have this medicament and show that this medicament can be as the inhibitor of RASGEF1A gene, illustrate that this medicament has the possibility that suppresses the cancer purposes, thereby conduct is used for the treatment of or candidate's medicament of preventing cancer.

Can assess the expression of gene level by method well-known in the art.For example, can follow under the title above ' I-1. is used for diagnosing cancer or the tendentious method of cancer takes place ' described method and measure RASGEF1A expression of gene level.

The cell or the cell mass that are used for above-mentioned evaluation can be any cell or any cell mass, as long as it expresses the RASGEF1A gene.For example, this cell or cell mass can be or comprise the epithelial cell that derives from tissue.Perhaps, this cell or cell mass can be or comprise the immortalized cells that derives from cancerous cells, comprise deriving from ICC, bladder cancer, lung cancer, cancer of the stomach, colorectal carcinoma, prostate cancer, chronic myeloid leukemia, kidney, soft tissue sarcoma, carcinoma of the pancreas and seminomatous those immortalized cellses.The cell of expressing the RASGEF1A gene comprises the clone of for example setting up from cancer (for example SSP25).In addition, this cell or cell mass can be or comprise the cell of using the RASGEF1A gene transfection.

Method of the present invention is allowed the various above-mentioned substances of screening, is specially adapted to the screening function nucleic acid molecule, comprises sense-rna, siRNA etc.

III-2-2. use the screening method of RASGEF1A genetic transcription regulatory region

According on the other hand, the invention provides a kind of method, it may further comprise the steps:

A) will be subjected to reagent agent and the cells contacting that has wherein imported following carrier, the reporter gene that described carrier comprises RASGEF1A gene transcription regulatory region and expresses under this transcriptional regulatory district control;

B) expression or the activity of the described reporter gene of detection; And

C) select with do not have the condition that is subjected to the reagent agent under detected level compare the expression that reduces described reporter gene or actively be subjected to the reagent agent.

The reporter gene and the host cell that are fit to are well-known in the art.Can use RASGEF1A gene transcription regulatory region to prepare the needed report construction of screening, described transcriptional regulatory district can obtain from genomic library as the Nucleotide section that contains the transcriptional regulatory district based on the Nucleotide information of this gene.

The transcriptional regulatory district can be the promoter sequence of RASGEF1A gene for example.

When using the reporter gene cells transfected that can be operatively connected, can identify the medicament that suppresses or strengthen the RASGEF1A expression of gene by the expression level that detects the reporter gene product through adjusting sequence (for example promoter sequence) with the RASGEF1A gene.

As reporter gene, for example Ade2 gene, lacZ gene, CAT gene, luciferase genes, HIS3 gene and such gene well-known in the art can be used.The method that is used to detect these expression of gene is well-known in the art.

III-3. select to be suitable for the therapeutical agent of particular individual

The difference that idiogenetics is formed can cause the relative capacity of the various medicines of its metabolism difference to occur.Thereby play the medicament of antineoplastic agent effect at the subject intracellular metabolite, can be by in experimenter's cell, inducing carcinous status flag genetic expression pattern to the transformation of non-carcinous status flag genetic expression pattern and oneself displays.Therefore, RASGEF1A gene at differential expression between cancerous cells and the non-cancerous cells disclosed here is allowed cancer therapy or the preventative inhibitor that test is inferred in from selected experimenter's population of test, thereby determines whether this medicament is suitable cancer inhibitor in this experimenter.

For evaluation is suitable for the cancer inhibitor of particular subject, will be exposed to candidate therapeutic agent from this experimenter's population of test, and measure the RASGEF1A expression of gene.

In the context of the inventive method, population of test contains the cancer cells of expressing the RASGEF1A gene.Preferably, subject cell is an epithelial cell.

Specifically, can there be incubation population of test under the condition of candidate therapeutic agent, measuring RASGEF1A expression of gene in the population of test, and compare with one or more contrast preface types, for example carcinous with reference to expressing preface type or non-carcinous with reference to expressing the preface type.

The RASGEF1A expression of gene shows that with respect to the reduction with reference to cell mass that contains cancer cells this medicament has the treatment potentiality in the population of test.

IV. be used for the treatment of or the pharmaceutical composition of preventing cancer:

The medicament that sifts out by any screening method of the present invention, the antisense nucleic acid of RASGEF1A gene and siRNA, and suppress or the biologic activity of containment RASGEF1A expression of gene or RASGEF1A polypeptide, also suppress or destroy Cycle Regulation and cell proliferation at the antibody of RASGEF1A polypeptide.Therefore, the invention provides and be used for the treatment of or the composition of preventing cancer, said composition comprises the antisense nucleic acid of the medicament that sifts out by any screening method of the present invention, RASGEF1A gene and siRNA or at the antibody of RASGEF1A polypeptide.Composition of the present invention can be used for treatment or preventing cancer, such as ICC, bladder cancer, lung cancer, cancer of the stomach, colorectal carcinoma, prostate cancer, chronic myeloid leukemia, kidney, soft tissue sarcoma, carcinoma of the pancreas and spermocytoma.

Said composition can be used as and is used for people or other mammiferous medicines, described Mammals such as mouse, rat, cavy, rabbit, cat, dog, sheep, pig, ox, monkey, baboon and chimpanzee.

In the context of the present invention, the pharmaceutical dosage form that is suitable for the activeconstituents of the present invention that details are as follows (comprising the medicament that sifted out, antisense nucleic acid, siRNA, antibody etc.) comprises and is suitable for that oral, rectum, nose, part (comprising oral cavity and hypogloeeis), vagina or parenteral (comprising intramuscular, subcutaneous and intravenously) use, or is suitable for by sucking or be blown into the pharmaceutical dosage form of using.Preferably, use by intravenously.Preparation is optional with discrete dose unit packing.

Be suitable for comprising capsule, microcapsule, cachet and the tablet of each self-contained predetermined amount activeconstituents for Orally administered pharmaceutical dosage form.The formulation that is fit to also comprises pulvis, elixir, granula, solution, suspension and emulsion.Activeconstituents can be chosen wantonly with the form of bolus (bolus), electuary (electuary) or paste (paste) and use.Perhaps, optionally, pharmaceutical composition can sterile solution or is non-Orally administered with the form of the suspension injection of water or any other pharmaceutically acceptable liquid.For example, activeconstituents of the present invention can be mixed with pharmaceutically acceptable carrier or medium, specifically, be sterilized water, physiological saline, vegetables oil, emulsifying agent, suspending agent, tensio-active agent, stablizer, perfume compound, vehicle, vehicle, sanitas, tackiness agent or the like, exist with the desired unit dosage form of common acceptable drug compound method.The amount that is contained in the activeconstituents in the above-mentioned preparation is the suitable dose in the obtainable stated limit.

The example that can mix the additive in tablet and the capsule includes but not limited to: tackiness agent, such as gelatin, W-Gum, tragacanth gum and gum arabic; Vehicle is such as crystalline cellulose; Swelling agent is such as W-Gum, gelatin and Lalgine; Lubricant is such as Magnesium Stearate; Sweeting agent is such as sucrose, lactose or asccharin; And perfume compound, such as spearmint oil, Bai Zhu (Gaultheria adenothrix) oil and cherry.Tablet can be by compression or molded the preparation, wherein optional one or more system component that contains.Compressed tablets can prepare by the following method: activeconstituents is chosen wantonly and tackiness agent, lubricant, inert diluent, lubricant, tensio-active agent or dispersant with free-pouring form (such as powder or particle), and suppressed in suitable machine.Molded tablet can prepare by the following method: carry out molded to the mixture that utilizes the moistening powder compounds of inert liquid diluent in suitable machine.Described tablet can carry out dressing according to method well-known in the art.Tablet can be chosen preparation wantonly so that activeconstituents slowly-releasing or controlled release in vivo to be provided.Can comprise a slice medicine of taking in every month in the packing of tablet.

In addition, when unit dosage form is capsule, outside mentioned component, also can comprise liquid vehicle, such as oil.

Oral liquid can form for example moisture or oleaginous suspension, solution, emulsion, syrup or elixir exist, or can the desciccate existence be used for rebuilding with water or other vehicle that is fit to before use.The aforesaid liquid preparation can contain conventional additives, such as suspending agent, emulsifying agent, non-water vehicle (can comprise edible oils) or sanitas.

The preparation that is used for parenteral administration comprises water-based and non-aqueous aseptic injectable solution, and it can contain antioxidant, buffer reagent, fungistat and make preparation and target receptor's the isoosmotic solute of blood; And water-based and non-aqueous sterile suspensions, it can contain suspending agent and thickening material.Described preparation can unitary dose or multi-dose container provide, for example Mi Feng ampoule and tubule can also be preserved under lyophilize (freeze-drying) condition, so only need to face to add sterile liquid carrier, for example salt solution, water for injection before using.Perhaps, can be provided for the preparation of continuous infusion.Join promptly that promptly the injection solution and the suspension of usefulness can be by sterile powder, particle and the tablet preparation of aforementioned type.

In addition, use the vehicle that is suitable for injecting, can prepare the aseptic mixture of injection according to common method for preparation of drug such as distilled water.Physiological saline, glucose and other isotonic liquid comprise adjuvant, can be used as the aqueous solution of injection such as D-sorbyl alcohol, D-seminose, D-N.F,USP MANNITOL and sodium-chlor.They can with appropriate solubilizing agent, such as alcohol, ethanol for example; Polyvalent alcohol is such as propylene glycol and polyoxyethylene glycol; And nonionic surface active agent, use together such as Polysorbate 80 (TM) and HCO-50.

Sesame oil or soybean oil can be used as oleaginous fluid, can be used in combination as solubilizing agent with peruscabin or phenylcarbinol, also can with damping fluid, such as phosphate buffered saline buffer and sodium acetate buffer; Analgesic agent is such as vovocan; Stablizer is such as phenylcarbinol and phenol; And/or antioxidant is prepared together.The injection of preparation can be encased in the suitable ampoule.

The preparation that is used for rectal administration comprises the suppository with standard vector such as theobroma oil or polyoxyethylene glycol.Be used for a mouthful interior topical, for example the preparation of oral cavity or sublingual administration comprises lozenge and pastille, wherein lozenge comprises activeconstituents in flavoured base such as sucrose and gum arabic or tragacanth gum, and pastille comprises activeconstituents in matrix such as gelatin, glycerine, sucrose or gum arabic.For the intranasal administration activeconstituents, but can use the liquid spray dispersion powder, or use with the form of drops.Drops can also contain one or more dispersion agents, solubilizing agent or suspending agent with water-based or the preparation of non-aqueous matrix in this matrix.

For by inhalation, the convenient means that can send aerosol spray by insufflator, spraying gun, pressurized package (pressurized pack) or other is delivering compositions easily.Pressurized package can contain suitable propelling agent, such as Refrigerant 12, trichlorofluoromethane, and dichloro tetrafluoro ethane, carbonic acid gas or other suitable gas.Under the situation of pressurized aerosol, can determine dose unit by the valve of sending metered amounts is provided.

Perhaps, for by sucking or being blown into administration, composition can be taked the form of dry powder composite, for example the powdered mixture of activeconstituents and the powder matrix (such as lactose or starch) that is fit to.Powder composition can provide by unit dosage form, and for example capsule, cartridge case (cartridge), gel (gelatin) or Blister Package (blister pack) by sucker or insufflator, can be used described powder by above-mentioned unit dosage form.

Other preparation comprises the implantable device and the adhesive patches that can discharge therapeutical agent.

When needing, can adopt the above-mentioned preparation that is suitable for slow-release.Can also contain other activeconstituents in the pharmaceutical composition, such as biocide, immunosuppressor or sanitas.Should be appreciated that outside the above-mentioned composition of specifically mentioning preparation of the present invention can contain this area for described preparation type other medicament commonly used, the preparation that for example is suitable for oral administration can contain seasonings.

Preferred unit dose formulations is those preparations that contain the effective dose of every kind of activeconstituents of the present invention described in the title hereinafter method of preventing cancer ' be used for the treatment of or ' or its suitable part.

IV-1. the pharmaceutical composition that comprises the medicament that is sifted out

The invention provides and comprise any medicament of filtering out by the invention described above screening method, be used for the treatment of or the composition of preventing cancer.

The medicament that filters out by the inventive method can directly be used or be formulated as dosage form according to any conventional medicine preparation method of above detailed description.

IV-2. the pharmaceutical composition that comprises siRNA

Can be used for reducing this expression of gene level at the siRNA of RASGEF1A gene (below be also referred to as " RASGEF1A siRNA ").At this, term " siRNA " refers to stop the double stranded rna molecule of said target mrna translation.In the context of the present invention, siRNA comprises at what raise marker gene RASGEF1A phosphorothioate odn sequence and an anti sense nucleotide sequence.Make up this siRNA and make it comprise adopted and the complementary antisense sequences of having of target gene, promptly comprise the Nucleotide of hairpin structure.

The siRNA of RASGEF1A gene and said target mrna hybridization promptly combine and therefore disturb mRNA to translate with normal strand mRNA transcript, finally reduce or suppress the generation (expression) of this genes encoding polypeptide.Thereby siRNA molecule of the present invention can be limited with the ability of the mRNA specific hybrid of RASGEF1A gene under stringent condition by it.

In the context of the present invention, the length of siRNA preferably is less than 500,200,100,50 or 25 Nucleotide.Preferred, the length of siRNA is 19-25 Nucleotide.The target nucleic acid sequence of exemplary RASGEF1AsiRNA comprises the nucleotide sequence of SEQ ID NO:9 or 13.In the RNA or derivatives thereof, the Nucleotide in the sequence " t " should be " u " and replaces.Therefore, for example, the invention provides comprise nucleotide sequence 5 '-double stranded rna molecule of gagaauggcacagugaaga-3 ' (SEQ ID NO:9) or 5 '-caucuacuuccugcacaaa-3 ' (SEQ ID NO:13).In order to strengthen the inhibition activity of siRNA, Nucleotide " u " can be added to 3 ' end of the antisense strand of target sequence.The number of " u " that adds is at least 2, is generally 2-10, preferred 2-5." u " that is added is at 3 ' the terminal strand that forms of the antisense strand of siRNA.

The ring sequence of being made up of any nucleotide sequence can be positioned between adopted sequence and the antisense sequences, so that form hairpin ring structure.Therefore, the present invention also provides has general formula 5 '-siRNA of [A]-[B]-[A ']-3 ', wherein [A] be corresponding to the ribonucleoside acid sequence of the sequence of the mRNA of RASGEF1A gene or cDNA specific hybrid.In preferred embodiments, [A] is the ribonucleoside acid sequence corresponding to the sequence of RASGEF1A gene; [B] is the ribonucleoside acid sequence of being made up of 3-23 Nucleotide; And the ribonucleoside acid sequence that the complementary sequence of [A '] serve as reasons [A] is formed.[A] district and the hybridization of [A '] district form the ring of being made up of [B] district then.The length of ring sequence can be preferably 3-23 Nucleotide.The ring sequence is for example optional from following sequence (http://www.ambion.com/techlib/tb/tb_506.html):

CCC, CCACC or CCACACC:Jacque JM et al., Nature 2002,418:435-8.

UUCG：Lee?NS?et?al.，Nature?Biotechnology?2002，20：500-5；Fruscoloni?Pet?al.，Proc?Natl?Acad?Sci?USA?2003，100(4)：1639-44。

UUCAAGAGA：Dykxhoorn?DM?et?al.，Nature?Reviews?Molecular?CellBiology?2003，4：457-67。

" UUCAAGAGA (being " ttcaagaga " in DNA) " is particularly suitable ring sequence.In addition, the ring sequence of being made up of 23 Nucleotide also provides activated siRNA (Jacque J-M et al., Nature 2002,418:435-8).

Be applicable to that the illustrative hair clip siRNA in the context of the invention comprises, for RASGEF1A-siRNA,

5 '-gagaauggcacagugaaga-[B]-ucuucacugugccauucuc-3 ' (target sequence that is used for SEQ ID NO:9); With

5 '-caucuacuuccugcacaaa-[B]-uuugugcaggaaguagaug-3 ' (target sequence that is used for SEQ ID NO:13).

Can use can derive from the Ambion website ( Http:// www.ambion.com/techlib/misc/SiRNA_finder.html) nucleotide sequence of the siRNA that siRNA designing computer programs design is fit to.This computer program is used for siRNA synthetic nucleotide sequence based on following process selecting.

The selection of siRNA target site:

1. the AUG initiator codon from the target transcript begins to scan downstream, seeks AA dinucleotides sequence.Write down 19 contiguous Nucleotide of the appearance of each AA and 3 ' side thereof as potential siRNA target site.Tuschl et al.Genes Cev 1999,13 (24): 3191-7, do not recommend zone (within 75 bases) design siRNA, because aforementioned region may be rich in the adjusting protein binding site at 5 ' and 3 ' non-translational region (UTR) and contiguous initiator codon.Conjugated protein and/or the translation initiation complex of UTR can disturb the combination of siRNA endonuclease enzyme complex.

2. described potential target site and people's gene group database are compared, do not consider and any target sequence of the remarkable homologous of other encoding sequence.Can adopt the BLAST (Altschul SF et al., Nucleic AcidsRes 1997, the 25:3389-402 that are found in NCBI server: www.ncbi.nlm.nih.gov/BLAST/; J Mol Biol 1990 215:403-10) carries out the homology search.

3. select qualified target sequence to be used to synthesize.At Ambion, can preferably select several target sequences along the length of gene to be assessed.

Can use the standard technique that is used for the siRNA transfered cell.For example, can with mRNA transcript bonded form with the direct transfered cell of the siRNA of RASGEF1A.In these embodiments, siRNA molecule of the present invention carries out as mentioned above the modification about antisense molecule usually.Also may carry out other modifications, for example, the pharmacological property that coupling has the siRNA of cholesterol to demonstrate to have improvement (Song et al., Nature Med 2003,9:347-51).

Perhaps, the DNA portability of coding siRNA is in carrier (below be also referred to as " siRNA carrier ").Can be operatively connected in the expression vector of adjusting sequence (for example from small nuclear rna (snRNA) U6 rna plymerase iii transcription unit or people H1RNA promotor) by for example target RASGEF1A gene order being cloned at this sequence flank, all express the mode of (by transcribing of this dna molecular) and produce above-mentioned carrier (Lee NS et al. to allow two chains, Nature Biotechnology 2002,20:500-5).For example, transcribe RNA molecule with the mRNA antisense of RASGEF1A gene by first promotor (promoter sequence of the DNA 3 ' side of for example being cloned), transcribing for the mRNA of RASGEF1A gene by second promotor (promoter sequence of the DNA5 ' side of for example being cloned) is the RNA molecule of sense strand.Sense strand and antisense strand are hybridized the siRNA construction that is used for reticent RASGEF1A genetic expression with generation in vivo.Perhaps, can utilize two kinds of constructions to create the sense strand and the antisense strand of strand siRNA construction.In this case, have secondary structure for example the construction of hair clip produce as the single transcript that comprises adopted sequence of having of target gene and complementary antisense sequences.

Thereby the present invention is used for the treatment of or the pharmaceutical composition of preventing cancer comprises siRNA or expresses the carrier of siRNA in vivo.

For with siRNA carrier transfered cell, can use the transfection toughener.FuGENE6 (Rochediagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen) and Nucleofector (Wako pure Chemical) can be used as the transfection toughener.Therefore, pharmaceutical composition of the present invention can further comprise above-mentioned transfection toughener.

IV-3. the pharmaceutical composition that comprises antisense nucleic acid

Antisense nucleic acid corresponding to the RASGEF1A gene nucleotide series can be used for being reduced in this expression of gene level that raises in the multiple cancerous cells, has the purposes of treatment cancer, therefore is also contained among the present invention.Antisense nucleic acid works by combining with the nucleotide sequence of RASGEF1A gene or its corresponding mRNA, thereby suppresses this gene transcription or translation, promotes the degraded of mRNA, and/or suppresses this coded by said gene protein expression.Therefore, the result is that antisense nucleic acid suppresses RASGEF1A albumen and bring into play function in cancerous cells.At this, phrase " antisense nucleic acid " refers to the Nucleotide with the target sequence specific hybrid, not only comprises and the complete complementary Nucleotide of this target sequence, also comprise comprise one or more Nucleotide mispairing, with the Nucleotide of this target complement sequence.For example, antisense nucleic acid of the present invention be included in have on the span of at least 15 continuous nucleotides of RASGEF1A gene or its complementary sequence at least 70% or higher, preferred at least 80% or higher, more preferably at least 90% or higher even more preferably at least 95% or the polynucleotide of higher homology.Algorithm as known in the art can be used for measuring above-mentioned homology.

Antisense nucleic acid of the present invention is by combining with the DNA or the mRNA of RASGEF1A gene, act on and produce the proteinic cell of this coded by said gene, suppress transcribing or translating of they, promote the degraded of mRNA, and suppress this protein expression, finally suppress this protein performance function.

By mixing, antisense nucleic acid of the present invention can be prepared as external preparation, for example liniment (liniment) or net for catching birds or fish agent (poultice) with suitable substrate material to the nucleic acid non-activity.

Equally, as required, by adding vehicle, isotonic agent, solubilizing agent, stablizer, sanitas, analgesic agent etc., antisense nucleic acid of the present invention can be mixed with tablet, powder, particle, capsule, liposome methods, injection, solution, nasal drop and lyophilized preparation.Antisense sealing medium (antisense-mountingmedium) also can be used for increasing weather resistance and membrane permeability.The example of described antisense sealing medium includes but not limited to liposome, poly-L-Methionin, lipid, cholesterol, lipofection agent or their derivative.Can prepare them by following currently known methods.

Antisense nucleic acid of the present invention suppresses the proteic expression of RASGEF1A and has the active purposes of this protein biology of inhibition.In addition, the expression inhibitor that comprises antisense nucleic acid of the present invention is useful, because they can suppress the proteic biologic activity of RASGEF1A.

Antisense nucleic acid of the present invention comprises the oligonucleotide through modifying.For example, sulfo-(thioated) oligonucleotide can be used to give oligonucleotide with the nuclease resistance.

IV-4. the pharmaceutical composition that comprises antibody

Can be suppressed at the function of crossing the gene product of the RASGEF1A gene of expressing in the multiple cancer by using following compound, described compound can combine or otherwise suppress its function with this gene product.Can be used as such compound at the antibody of RASGEF1A polypeptide and be mentioned, and can be used as and be used for the treatment of or the active ingredient in pharmaceutical of preventing cancer.

The present invention relates to segmental application at proteinic antibody or this antibody of RASGEF1A coded by said gene.As used herein, term " antibody " refer to have ad hoc structure, only with the antigen that is used for synthetic this antibody (promptly raising the gene product of sign) or with the immunoglobulin molecules of the closely-related AI of this antibody (promptly combining).The molecule that comprises the antigenic molecule that is used for synthetic antibody and be included as this antigenic epi-position that antibody discerns can be used as with the closely-related antigen of this antibody and is mentioned.

In addition, the antibody that is used for pharmaceutical composition of the present invention can be antibody fragment or the antibody through modifying, as long as the protein bound (the immunologic competence fragment of for example anti-RASGEF1A antibody) of itself and RASGEF1A coded by said gene.For instance, antibody fragment can be Fab, F (ab ') ₂, Fv or strand Fv (scFv), wherein from the Fv fragment of H and L chain by suitable joint be connected (Huston JS et al., Proc Natl Acad Sci USA 1988,85:5879-83).Can be by producing such antibody fragment with enzyme such as papoid or pepsin antibody.Perhaps, can make up the segmental gene of encoding antibody, be inserted into expression vector, and express in suitable host cells that (referring to for example Co MS et al., JImmunol 1994,152:2968-76; Better M et al., Methods Enzymol 1989,178:476-96; Pluckthun A et al., Methods Enzymol 1989,178:497-515; Lamoyi E, Methods Enzymol 1986,121:652-63; Rousseaux J et al., Methods Enzymol 1986,121:663-9; Bird RE et al., Trends Biotechnol 1991,9:132-7).

Can be by coming modified antibodies, such as polyoxyethylene glycol (PEG) with multiple molecule coupling.The present invention includes such antibody through modifying.Can obtain the antibody that process is modified by chemically modified antibody.Above-mentioned modifying method is conventional in this area.

Perhaps, be used for antibody of the present invention and can be having and derive from the non-human antibody's of RASGEF1A polypeptide variable region and derive from the chimeric antibody of the constant region of people's antibody, or comprise the complementary determining region (CDR) that derives from the non-human antibody, the framework region (FR) that derives from people's antibody and the humanized antibody of constant region.Can use known technology to prepare above-mentioned antibody.Can (referring to as Verhoeyen et al., Science 1988,239:1534-6) by carry out humanization with the corresponding sequence of the CDR of rodent or CDR sequence replacing people antibody.Thereby such humanized antibody is a chimeric antibody, is wherein substituted by the corresponding sequence from inhuman species less than whole person's variable domain basically.

The whole person's antibody that also comprises the people variable region outside people's framework region and constant region also is utilizable.Such antibody can utilize the various techniques known in the art preparation.For example, in vitro method comprise people's antibody fragment that use is showed on phage the reorganization library (Hoogenboom et al. for example, J Mol Biol1992,227:381).Similarly, can by human immunoglobulin gene's seat is introduced for example endogenous immunoglobulin gene of transgenic animal partially or completely the mouse of deactivation prepare people's antibody.This method is described in for example United States Patent(USP) Nos. 6,150,584; 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; With 5,661,016.

In the time the antibody that is obtained will being applied to human body (Antybody therapy), preferred people's antibody or humanized antibody are because they have the immunogenicity of reduction.

Can be with the antibody purification that as above obtains to homogeneous.For example, can carry out separating of antibody and purifying with purification process according to the separation that is used for usual protein.For example, by suitable selection and be used in combination column chromatography such as affinity chromatography, filtration, ultrafiltration, saltout, dialysis, sds polyacrylamide gel electrophoresis, isoelectrofocusing etc., can separate and separation antibody (Antibodies:A Laboratory Manual.Ed Harlowand David Lane, but be not limited thereto Cold Spring Harbor Laboratory (1988)).Albumin A post and Protein G post can be used as affinity column.Exemplary spendable albumin A post comprises for example Hyper D, POROS and Sepharose F.F. (Pharmacia).

Except that affinity chromatography, exemplary chromatography for example comprises: (Strategies for Protein Purification andCharacterization:A Laboratory Course Manual.Ed Daniel R.Marshak et al., Cold Spring Harbor Laboratory Press (1996)) such as ion exchange chromatography, hydrophobic chromatography, gel-filtration, reversed phase chromatography, adsorption chromatographies.Can pass through liquid chromatography (LC), carry out the chromatography operation such as HPLC and FPLC.

V. be used for the treatment of or the method for preventing cancer:

The cancer therapy that changes at the specific molecular that takes place in the cancer cells is identified as effectively by the clinical development of following antitumor drug and the approval of administration: such as the trastuzumab that is used for the treatment of terminal cancer (trastuzumab) (Herceptin), the imatinib mesylate (imatinib methylate) that is used for the treatment of chronic myeloid leukemia (Gleevec), the gefitinib (Iressa) that is used for the treatment of nonsmall-cell lung cancer (NSCLC), and be used for the treatment of Rituximab (rituximab) (anti-CD20mAb) (the Ciardiello F et al. of B cell lymphoma and lymphoma mantle cell, Clin Cancer Res 2001,7:2958-70, Review; Slamon DJ et al., N Engl J Med 2001,344:783-92; Rehwald Uet al., Blood 2003,101:420-4; Fang G et al., Blood 2000,96:2246-53).Be effectively on these clinical drugs, and have better tolerance, because their target transformants only than traditional antineoplastic agent.Therefore, these medicines have not only improved cancer patients's survival rate and quality of life, and have verified the theory of molecular targeted cancer therapy.In addition, when targeted drug and standard chemotherapy are used in combination, can strengthen the standard chemotherapy effectiveness (Gianni L, Oncology 2002,63Suppl 1:47-56; Klejman A et al., Oncogene 2002,21:5868-76).Therefore, Wei Lai cancer therapy will relate to probably with conventional medicine and the target-specific medicament combination that takes place at tumour cell different qualities such as blood vessel and invade.

These control methods can exsomatize (ex vivo), in external (for example by culturing cell in the presence of medicament) or the body (for example by using medicament) to the experimenter carry out.Described method comprises the combination of administration of protein or proteinic combination or nucleic acid molecule or nucleic acid molecule, as the therapy of the abnormal activity of the unconventionality expression that resists difference expression gene or its gene product.

Can treat disease and the illness that the expression level that is characterised in that gene and gene product or biologic activity (with respect to the experimenter who does not suffer from disease or illness) raise respectively to some extent with the active therapeutical agent that antagonism (promptly reduce or suppress) is crossed expressing gene.Can therapeutic or the preventative therapeutical agent of using antagonistic activity.

Therefore, available therapeutical agent for example comprises that (i) crosses polypeptide or its analogue, derivative, fragment or the homologue of the RASGEF1A gene of expressing in the context of the invention; (ii) at the gene of crossing expression or the antibody of gene product; (iii) the encoded nucleic acid of the gene of expressing; The antisense nucleic acid or the nucleic acid of (iv) " dysfunction " (promptly because due to the allos insertion in the nucleic acid of mistake expressing gene); (v) siRNA (siRNA); Or (vi) conditioning agent (promptly changing interactional inhibitor, antagonist between polypeptide expressed and its binding partners).The antisense molecule of dysfunction is used to the endogenous function of " knocking out " polypeptide by homologous recombination, and (referring to for example Capecchi, Science 1989,244:1288-92).

By obtaining patient tissue samples (for example from biopsy) and in the structure and/or the activity of its RNA of external test or peptide level, expressed the peptide mRNA of the gene that changes to some extent (or express), quantitation of peptides and/or RNA, thus can be easy to the rising of the level of detecting.Method well known in the art includes but not limited to immunoassay (for example Western engram analysis, immunoprecipitation are followed sodium lauryl sulphate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry etc.) and/or is used to detect the hybridization assays method (for example Northern trace assay method, Dot blot, in situ hybridization etc.) that mRNA expresses.

Before the obvious clinical symptom of disease occurs, carry out preventive administration, make disease or illness be prevented or postpone its development.

Methods of treatment of the present invention can comprise the step that cell is contacted with one or more active medicaments of regulating the RASGEF1A gene product.The example of regulating the medicament of protein active includes but not limited to nucleic acid, protein, the related part of above-mentioned proteinic natural existence, peptide, peptide mimics and other small molecules.

Therefore, the invention provides the method for activity by reducing RASGEF1A expression of gene or this gene product treatment or relieving cancer symptoms or preventing cancer in the experimenter.Method of the present invention is specially adapted to treatment or prevention ICC, bladder cancer, lung cancer, cancer of the stomach, colorectal carcinoma, prostate cancer, chronic myeloid leukemia, kidney, soft tissue sarcoma, carcinoma of the pancreas and spermocytoma.

The therapeutical agent that preventability or therapeutic administration are fit to is given the experimenter who suffers from cancer or risky generation cancer (or suspecting the generation cancer).Can be by using the standard clinical method or identifying such experimenter by detecting the RASGEF1A gene abnormal expression level (" rise " or " cross and express ") or the abnormal activity of this gene product.

According to an aspect of the present invention, the medicament by the inventive method screening can be used for treatment or preventing cancer.Can use the well-known method of those skilled in the art to give the patient with these pharmacy applications, for example, in intra-arterial, intravenously or percutaneous injection or the nose, through segmental bronchus, intramuscular or oral administration.Can encode if described medicament is DNA, then the DNA insertion can be used for the carrier of gene therapy, and use this carrier and give the patient to implement this therapy.

Body weight, age, sex, symptom, situation and medication according to patient to be treated change dosage and method; Yet those skilled in the art can conventionally select proper dosage and medication.

For example, depend on above-mentioned multiple factor although combine and regulate the dosage of the medicament of this polypeptide active with the RASGEF1A polypeptide, when normal type grownup (60kg) oral administration, this dosage be generally about 0.1mg to about 100mg every day, preferably about 1.0mg arrives about 50mg every day, more preferably from about 1.0mg arrives about 20mg every day.

When with the injection form during to normal type grownup (60kg) administered parenterally, although have some differences according to patient, target organ, symptom and medication, the about 0.01mg of intravenous injection is to about 30mg every day, preferably about 0.1mg is to about 20mg every day, more preferably from about 0.1mg is suitable to the dosage of about 10mg every day.Under the situation of other animal, can calculate proper dosage by being converted to 60kg body weight routine.

Similarly, pharmaceutical composition of the present invention can be used for treatment or preventing cancer.Can use the well-known method of those skilled in the art that described composition is applied to the patient, for example in intra-arterial, intravenously or percutaneous injection or the nose, through segmental bronchus, intramuscular or oral administration.

For aforementioned each situation, can the about 250mg/kg of about 0.1-the dosage range of every day oral or come applying said compositions, for example polypeptide and organic compound by injection.Adult's dosage range be generally about 5mg to about 17.5g/ days, preferably about 5mg arrives about 10g/ days, most preferably from about 100mg arrives about 3g/ days.Tablet or other unit dosage form that provides with discrete unit can contain easily with such dosage or as a plurality of such dosage effectively to be measured, for example contain the 5mg that has an appointment to about 500mg, be generally about 100mg to about 500mg.

Used dosage depends on multiple factor, comprises experimenter's age, body weight and sex, the definite illness and the severity thereof of being treated.In addition, route of administration also changes with illness and severity thereof.Yet in any case, those skilled in the art can both consider conventionally on the basis of above-mentioned factor to calculate suitable and dosage the best.

Specifically, can make it in the blood vessel to arrive ailing position by will directly being applied to ailing position at the antisense nucleic acid of RASGEF1A gene or being injected into, thereby be applied to the patient.

According to status of patient, the dosage of antisense nucleic acid derivative of the present invention can be done suitably to regulate and use with aequum.For example, can use the dosage range of 0.1-100mg/kg, preferred 0.1-50mg/kg.

VI. at the vaccine inoculation of cancer:

The present invention relates to a kind ofly be used for the treatment of or prevent method for cancer among the experimenter, the vaccine that comprises the immunologic competence fragment of using the polypeptide (being the RAS polypeptide) that comprises the RASGEF1A genes encoding, described polypeptide or coding aforementioned polypeptides or its segmental polynucleotide is in described experimenter's step.In the present invention, at the vaccine of cancer refer to the to have the ability material of in being inoculated into animal back inducing antitumor immunity power.

In the experimenter, use so vaccine-induced antineoplastic immune power.According to the present invention, RASGEF1A polypeptide and immunologic competence fragment thereof are considered to HLA-A24 or HLA-A*0201 restriction epitope peptide, and it can induce the strong and antigen-specific immune responses at the cancer cells of expressing the RASGEF1A gene.Therefore, the invention still further relates to a kind of by using the polypeptide (being the RAS polypeptide) that comprises the RASGEF1A genes encoding, described polypeptide the immunologic competence fragment or the vaccine of coding aforementioned polypeptides or its segmental polynucleotide in needed experimenter is arranged, be used for method in described experimenter's inducing antitumor immunity power.

These methods are specially adapted to treatment or prevention ICC, bladder cancer, lung cancer, cancer of the stomach, colorectal carcinoma, prostate cancer, chronic myeloid leukemia, kidney, soft tissue sarcoma, carcinoma of the pancreas and spermocytoma.

In some cases, RASGEF1A albumen or its immunologic competence fragment can be bonded to TXi Baoshouti (TCR) or be used by the form that APC such as scavenger cell, dendritic cell (DC), B cell or PBMC present.Because the strong antigen of DC is presented ability, therefore in APC, most preferably use DC.

Generally speaking, antineoplastic immune power comprises such as following immunne response:

-induce cytotoxic lymphocyte at tumour,

-induce identification tumour antibody and

-inducing antitumor production of cytokines.

Therefore, when inducing any these immunne responses behind certain protein inoculation animal, determine that this protein has antineoplastic immune power and induces effect.Any protein fragments of RASGEF1A polypeptide all can be used as the immunologic competence fragment of the inventive method.

Can by in vivo or the observation in vitro host immune system detect protein inducing at this proteinic replying to antineoplastic immune power.

It is for example, a kind of that to be used to detect CTL inductive method be well-known.Specifically, the foreign matter that enters live body is presented to T cell and B cell by the effect of APC.The T cell that responds to APC institute antigen-presenting in the antigen-specific mode is bred (this is called the T cell activation) then owing to this antigenic stimulation is divided into CTL.Therefore, induce can be by being presented to the T cell with this peptide via APC and detecting inducing of CTL and assess for the CTL of certain peptide.In addition, APC has the effect that activates CD4+T cell, CD8+T cell, scavenger cell, eosinocyte and NK cell.Because CD4+T cell and CD8+T cell also are important in antineoplastic immune power, can use the activation effect of these cells to assess the antineoplastic immune power inducing action of peptide as index.

A kind of DC of use is well known in the art as the method that APC assesses the CTL inducing action.In APC, DC is the representative APC with the strongest CTL inducing action.For whether the fragment of determining the RASGEF1A polypeptide has immunologic competence, and whether can be used for method of the present invention, at first will be tried polypeptide (being any fragment of RASGEF1A polypeptide) and contact, then with DC and T cells contacting with DC.With after DC contacts,, represent that then this is tried polypeptide and has inducing cytotoxic T cell activity if detect the T cell that has at the cytotoxic effect of target cell.CTL can for example adopt the dissolving of the tumour cell of 51Cr mark to detect as index at the activity of tumour.Perhaps, the method that adopts 3H-thymidine picked-up activity or LDH (lactose desaturase) release to assess the tumour cell degree of injury as index also is well-known.

Except that DC, PBMC also can be used as APC.Reported by under the situation that has GM-CSF and IL-4, cultivating PBMC and come inducing of enhanced CT L.Similarly, shown by there being the key hole

Cultivate PBMC under the condition of hemocyanin (KLH) and IL-7 and induce CTL.

Can think that the polypeptide that tried that has the CTL induced activity by these method confirmations is the polypeptide with DC activation effect and CTL induced activity subsequently.Therefore, can induce polypeptide at the CTL of tumour cell to can be used as vaccine at tumour.In addition, also can be used as vaccine by contact the APC that obtains to induce at the ability of the CTL of tumour with described polypeptide at tumour.In addition, presenting polypeptide antigen by APC obtains Cytotoxic CTL and also can be used as vaccine at tumour.Utilization is called the cellular immunization therapy by these tumor therapeuticing methods of the antineoplastic immune power that APC and CTL produce.

Generally speaking, when using polypeptide to be used for the cellular immunization therapy, known by make up multiple have the polypeptide of different structure and it contacted with DC improve CTL inductive efficient.Therefore, when stimulating DC with protein fragments, it is favourable using polytype segmental mixture.

Perhaps, polypeptide can be confirmed by observing at the inducing of antibody generation of tumour the inducing of immunizing power at tumour.For example, when inducing generation at the antibody of this polypeptide in the experimental animal with polypeptide immune, and when these antibody inhibiting tumor cells growths, described polypeptide can be considered the ability with inducing antitumor immunity power.

By using vaccine-induced antineoplastic immune power of the present invention, and inducing of this antineoplastic immune power makes treatment for cancer and prevention be able to possibility.Can comprise any following step at treatment for cancer or to the prevention of cancer outbreak: such as the inhibition of growth of cancer cells, the degeneration of cancer (involution), and the inhibition of oncogenesis.Treatment for cancer or prevention comprise also that the individual death rate of suffering from cancer and sickness rate reduce, the alleviation etc. of the detected symptom that the tumor markers level reduces, cancer occurs together in the blood.Above-mentioned therapeutic and prophylactic effects are preferably significant on the statistics.For example, under observation significance level is 5% or lower, wherein vaccine is compared at the therapeutic or the prophylactic effects of cell proliferation disorders and the contrast of not using vaccine.For example, StudentShi t check, Mann-Whitney U check or ANOVA can be used for statistical study.

Fragment or vectors encoding them that above-mentioned RASGEF1A polypeptide or its have immunologic competence can make up with adjuvant.Adjuvant refers to when strengthening compound at the immunne response of RASGEF1A polypeptide when using with polypeptide or fragment common (or in succession) with immunologic competence.Exemplary adjuvant includes but not limited to Toxins,exo-, cholera, Salmonellas toxin, alum etc.In addition, vaccine of the present invention that can suit with pharmaceutically acceptable carrier combinations.The example of such carrier comprises sterilized water, physiological saline, phosphate buffered saline buffer, nutrient solution etc.In addition, vaccine can comprise stablizer, suspension agent, sanitas, tensio-active agent etc. as required.But vaccine system or topical application.Vaccine administration can be undertaken by single-dose, perhaps strengthens by multiple dosing.

When using APC or CTL, can for example treat or prophylaxis of tumours by the method that exsomatizes as vaccine of the present invention.More specifically, can collect the experimenter's who receives treatment or prevent PBMC, cell be contacted under isolated condition with RASGEF1A polypeptide or its fragment, and after inducing APC or CTL, this cell is applied to the experimenter.Also can under isolated condition, import PBMC by carrier and induce APC coded polypeptide.External evoked APC or CTL can clone before using.Have high target cell by clone and cultivation and destroy active cell, can more effective enforcement cellular immunization therapy.In addition, not only can be used for carrying out the cellular immunization therapy, can also be used for carrying out the cellular immunization therapy at tumour from other individual similar type at the experimenter that cell is provided with isolating APC of this mode and CTL.

In addition, provide the pharmaceutical composition that is used for the treatment of or prevents cell proliferation disorders such as cancer, it comprises pharmaceutically RASGEF1A polypeptide or its immunologic competence fragment of significant quantity.Such pharmaceutical composition can be used for producing antineoplastic immune power.

Hereinafter, with reference to embodiment, the present invention will obtain more detailed description.Yet following material, method and embodiment are only illustrating each side of the present invention, never are intended to limit the scope of the invention.Therefore, can be used for practice of the present invention or test with similar or suitable method described herein and material.

Embodiment

I. material and method

1. clone and tissue sample

Monkey-kidney cells is that (American Type Culture Collection ATCC) locates to obtain from American type culture collection for COS7 and l cell clone NIH3T3.People's cholangiocarcinoma cell is that (Japanese Collection of ResearchBioresources JCRB) locates to obtain SSP25 from Japanology Biological resources collecting center.All cells is cultivated as cell monolayer in following suitable culture medium: be used for COS7 and NIH3T3 DullbeccoShi improvement EagleShi substratum (Sigma-AldrichCorporation, St.Louis, MO); And be used for SSP25 RPMI 1640 (Sigma-AldrichCorporation, St.Louis, MO).Clinical tissue derives from patient's informed consent, that accept hepatectomy specimens from pri.

2.RNA preparation and sxemiquantitative RT-PCR

The inventor uses the laser microbeam micromanipulative technique to collect from the cholangiocarcinoma cell of specimens from pri and the pure cell mass of non-carcinous bile duct epithelial cell.Cutting into slices as previously mentioned, preparation, laser microbeam micro-dissection, total RNA extract, (Nakamura T et al., Oncogene 2004,23:2385-400) based on the amplification of T7.Preparation from the epithelial mixture of normal stones in intrahepatic bile duct of 10 no ICC patient's hepatic tissues as general contrast.According to manufacturers protocol, utilize TRIZOL reagent (Invitrogen) from culturing cell, to extract total RNA.Use Superscript II reversed transcriptive enzyme (Life Technologies), utilize poly-dT _12-18Primer (Amersham Biosciences) is strand cDNA with the RNA reverse transcription that is extracted.Every part of strand cDNA dilution is used for pcr amplification afterwards.In GeneAmp PCR system 9700 (Perkin-Elmer), the RT-PCR that carries out standard in the PCR damping fluid (TAKARA) of 12 μ l volumes is with amplification, i.e. 94 ℃ of sex change 4 minutes, carry out 25 (for ACTB) or 30 (for RASGEF1A) circulation afterwards: 94 ℃ continue 20 seconds, 53 ℃ (for ACTB) or 60 ℃ (for RASGEF1A) continues 30 seconds, and 72 ℃ continue 30 seconds.Primer sequence is as follows; Be used for 5 of ACTB '-CAAGATGAGATTGGCATGG-3 ' (SEQ ID NO:3) and 5 '-TCTCCTTAGAGAGAAGTGG-3 ' (SEQ ID NO:4); And be used for 5 of RASGEF1A '-TTTGCACTTTCTGAGGTTCTAGC-3 ' (SEQ ID NO:5) and 5 '-GTCCTGCATTATGGTACAGCTTC-3 ' (SEQ ID NO:6).

3.Northern engram analysis

With the people organize more RNA trace (Clontech) with ³²The RASGEF1A cDNA of P mark is hybridized.Carry out prehybridization, hybridization and washing according to manufacturer's suggestion.With intensifying screen at-80 ℃ to trace radioautograph 120 hours.

4.RASGEF1A Subcellular Localization

Complete coding region (Genbank registration number AK095136 (SEQ ID NO:1) by RT-PCR amplification RASGEF1A, the aminoacid sequence of coding SEQ ID NO:2), use as next organizes primer: 5 '-ATTGAATTCCGGCCAGAATGTTCCTGGAGC-3 ' (SEQ ID NO:7) and 5 '-AATCTCGAGGGCTCTGTTCAGAAGGGTGGTC-3 ' (SEQ ID NO:8), and it is cloned into the suitable enzyme site (pCAGGS-n3Fc-RASGEF1A) of pCAGGS-n3Fc carrier.Will be to the cavate slide glass with the NIH3T3 cell transfer of the plasmid transient transfection of the RASGEF1A that expresses band FLAG label.After the transfection 24 hours, with the fixing culturing cell 15 minutes of the PBS that contains 4% paraformaldehyde, making cell have permeability in 2.5 minutes with the PBS processing that contains 0.1%Triton X-100 then in room temperature.Cover cell 24 hours with the sealing non-specific hybridization at 4 ℃ with the PBS that contains 3%BSA, then with anti-FLAG antibody (SIGMAF-3165) incubation as first antibody.There is anti-mouse IgG second antibody (the ICN/Cappel and Jackson Immuno Research) antagonist of substrate to carry out fluorescent dye with coupling.With 4 ', 6-diamidino-2-benzene indoles two hydrochloric acid (DAPI) pair cell nuclear is redyed.Use TCS-SP2 spectrum confocal scanning system (Leica) to obtain fluoroscopic image.

5. colony forming assay method

With plasmid (pCAGGS-n3Fc-RASGEF1A) rotaring redyeing COS 7 cell of expressing RASGEF1A.With the cell of antisence RASGEF1A (pCAGGS-n3Fc-RASGEF1A (-)) or simulation plasmid (pCAGGS-n3Fc-Mock) and the Geneticin incubation of suitable concentration 14 days.Also use Jim Sa solution-dyed with 100% methyl alcohol fixed cell.According to the scheme of manufacturer, and use cell counting test kit (DOJINDO, kumamoto, Japan) analysis of cells viability, triplicate.

6. make up siRNA expression vector at RASGEF1A

By the BbsI site that double chain oligonucleotide is cloned into aforementioned psiH1BX3.0 carrier prepare the plasmid of expressing siRNA (Shimokawa T et al., Cancer Res 2003,63:6116-20).The sequence of paired oligonucleotide is as follows: be used for 5 ' of siRNA-B-TCCCGAGAATGGCACAGTGAAGATTCAAGAGATCTTCACTGTGCCATTCTC-3 ', (SEQ ID NO:10) and 5 '-AAAAGAGAATGGCACAGTGAAGATCTCTTGAATCTTCACTGTGCCATTCTC-3 ' (SEQ ID NO:11); Be used for 5 ' of siRNA-E-TCCCCATCTACTTCCTGCACAAATTCAAGAGATTTGTGCAGGAAGTAGATG-3 ' (SEQ ID NO:14) and 5 '-AAAACATCTACTTCCTGCACAAATCTCTTGAATTTGTGCAGGAAGTAGATG-3 ' (SEQ ID NO:15); And be used for 5 ' of siRNA-EGFP-TCCCGAAGCAGCACGACTTCTTCTTCAAGAGAGAAGAAGTCGTGCTGCTTC-3 ' (SEQ ID NO:18) and 5 '-AAAAGAAGCAGCACGACTTCTTCTCTCTTGAAGAAGAAGTCGTGCTGCTTC-3 ' (SEQ ID NO:19).After with T4 polynucleotide kinase phosphorylation, boiled the paired oligonucleotide 5 minutes, produce double chain oligonucleotide by slowly cooling off to anneal subsequently.After the transfection 24 hours, check the expression of RASGEF1A by sxemiquantitative RT-PCR.It is as follows to be used for siRNA sequence of the present invention.

Table 1

7. cell survival amylograph

Use Nucleofector (AMAXA) or FuGENE6 reagent (Roche Diagnostics), the SSP25 cell of on the 10cm plate, cultivating with the plasmid transfection of expression siRNA (1 * 10 ⁶Individual cell/plate).In being supplemented with the substratum that contains 10% foetal calf serum of 0.9mg/ml Geneticin, keep two weeks of transfectional cell.Cell with the fixing survival of 100% methyl alcohol is also used Jim Sa solution-dyed.In another test, (DOJINDO, Kumamoto Japan) measure the cell of surviving with the cell counting test kit.Be to analyze the effect that RASGEF1A is expressed, after the transfection 24 hours from the total RNA of cell extraction and carry out sxemiquantitative RT-PCR and analyze.

The assay method 8.Ras Nucleotide dissociates

The complete coding region of K-Ras (amino acid/11-188), Ha-Ras (amino acid/11-188), N-RAS (amino acid/11-189), RASGEF1A or Sos catalytic domain (amino acid 564-1049) is cloned into the suitable cloning site (BamHI and XhoI) of pGEX-6P bacterial expression vector (Amersham Pharmacia Biotech).In e. coli strain bl21, express gst fusion protein,, use gsh-Sepharose 4B resin (Amersham Pharmacia Biotech) purification of recombinant proteins from full cell pyrolysis liquid according to the scheme of manufacturer.As carry out as described in a separate paper Ras Nucleotide dissociate assay method (Hall BE et al., J BiolChem 2001,276:27629-37).By on the heating shaking table, will be in 40pmol Ras and 400pmol[8-in the 100ml damping fluid that contains 20mM Tris (pH8.0), 50mM NaCl, 1mg/ml bovine serum albumin(BSA) (BSA), 1mM dithiothreitol (DTT) and 1mM EDTA ³H] GDP (12.4Ci/mmol of mark; Amersham Biosciences), makes GST-Ras fusion rotein and [8-30 ℃ of incubations 15 minutes ³H] guanosine 5 '-bisphosphate (GDP) combination of mark.Then 30 ℃ with the 20ml mixture with contain or do not contain the further incubation of 20ml damping fluid 15 minutes of RASGEF1A of recombinating, described damping fluid contains 20mM Tris (pH8.0), 50mM NaCl, 1mg/ml BSA, 1mM dithiothreitol (DTT), 5mM MgCl ₂And 0.8mMGTPgS.With the Sos catalytic domain as positive control.By adding ice-cold stop buffer (20mM Tris (pH8.0), 50mM NaCl and the 15mM MgCl of 40ml ₂) come termination reaction, and will mix object point at P81 phosphorylated cotton side sheet (square) (Upstate; Catalogue #20-134) on.With the ice-cold stop buffer washed twice of 3ml, with filter membrane install on the Aloka LSC-5100 liquid scintillation counter with measurement be bonded to Ras [ ³H]-radioactivity of GDP.

9.Ras active analysis

According to the scheme of manufacturer, utilize Ras activate to measure test kit (Upstate, catalogue #17-218 batch of #26220), the activation that comes measure R as by Raf-1 " drop-down " assay method.Specifically, the preparation coupling has the sepharose 4B of gst fusion protein, and this gst fusion protein contains the RBD of Raf-1, and 4 ℃ with the lysate incubation of the COS7 cell of plasmid that expression RASGEF1A is arranged from transfection or control plasmid (stand-in) 45 minutes.Precipitation is bonded to the protein of Raf-1, and separates on SDS-PAGE, uses anti-general Ras antibody (Upstate) to analyze by the Western trace subsequently.

10. wound healing and Matrigel invade assay method

By RT-PCR amplification RASGEF1A (the GenBank registration number: complete coding region AK095136), use one group of primer: 5 '-ATTGAATTCCGGCCAGAATGTTCCTGGAGC-3 ' (SEQ ID NO:7) and 5 '-AATCTCGAGGGCTCTGTTCAGAAGGGTGGTC-3 ' (SEQ ID NO; 8), then it is cloned into the suitable enzyme site (pCAGGS-n3Fc-RASGEF1A) of pCAGGS-n3Fc carrier.By with pCAGGS-n3Fc-RASGEF1A or pCAGGS-Mock plasmid transfection COS7 cell, set up COS7 cell (COS7-RASGEF1A) and the control cells (COS7-Mock) of expressing RASGEF1A.Cell is maintained in the substratum that is supplemented with the 0.9mg/ml Geneticin, and two weeks were selected single colony after the transfection.Use anti-FLAG antibody to confirm the expression of RASGEF1A by the Western trace.According to the scheme of manufacturer, (Japan) analysis of cells is grown for DOJINDO, Kumamoto, and is triplicate to use the cell counting test kit.For the wound healing migration assay, culturing cell 2 days and is swiped in the cross mode on the monolayer cell that converges with the plastic suction pipet tip to converging in 6 orifice plates.Several surface of a wound are labeled direction, observe at fixed time after scraping and follow by phase contrast microscopy (phase-contact microscopy) and take a picture.Use has the BD BioCoat of 8 μ m holes ^TMMatrigel ^TM(BD Biosciences, San Jose CA) check the cell intrusion to invade the chamber.Behind the incubation 36 hours, in 5 independent visuals field, the COS7-RASGEF1A that passes the migration of this chamber and the number of COS7-Mock cell are counted at microscopically.Statistical study is measured significance,statistical by StudentShi t check.

II. result

1.RASGEF1A the expression in the various human cancer raises

Use contains the genome range cDNA microarray of 27,648 genes, and the inventor has analyzed the expression preface type of 25 routine ICC, and it expresses the frequent gene by filter that raises by setting in tumour to have identified 52.Outside these 52 genes, also find a kind of internal registration number for D4223, corresponding to the gene of the EST of Hs.125293 in state-run biotechnology information center (National Center for Biotechnology Information) the Unigene database (Build#183), it compares remarkable rise above twice in 14 examples by what set with normal stones in intrahepatic bile duct epithelial cell in 11 examples (78.6%) of the ICC of filter.In the microarray data that the inventor obtained, compare with corresponding non-carcinous contrast, D4223 is expressed in also to have raise in 8 examples (18.2%), all 5 routine kidneys (100%), all 5 routine soft tissue sarcomas (100%), all 3 routine carcinoma of the pancreas (100%) and all the 3 routine spermocytomas (100%) of 1 example (33.3%), 44 routine chronic myeloid leukemias of 3 examples (60%), the 3 routine prostate cancers of 4 examples (80%), the 5 routine colorectal carcinomas of 16 examples (61.5%), the 5 routine cancer of the stomach of 8 examples (88.9%), the 26 routine lung cancer of 9 routine bladder cancer and surpasses twice.Sxemiquantitative RT-PCR afterwards analyzes and has disclosed D4223 expression rising (Figure 1A) in 9 examples of the 13 routine ICC that carry out microarray analysis.Be the expression level of assessment D4223 in people's normal adult tissue, the inventor uses D4223cDNA to carry out organizing the Northern engram analysis as probe more, and identify the transcript of an about 3.3kb, its moderate in brain, spinal cord is expressed, weak expression in lymphoglandula, suprarenal gland, but in other 19 tissues of being checked without any expressing (Figure 1B).This gene is named as RASGEF1A after a while, because it contains the RAS-GEF structural domain.The homology search that the RASGEF1A aminoacid sequence that use is inferred carries out demonstrates it and PDZ-GEF2 has 30% identity, has 29% identity with PDZ-GEF1.

2.RASGEF1A Subcellular Localization

Be the Subcellular Localization of investigation RASGEF1A, the inventor has expressed the RASGEF1A (Fig. 2 B) of band FLAG label by the transfection of plasmid (p3Xflag-RASGEF1A) in the NIH3T3 cell, and carried out immunocytochemical stain.This immunocytochemical stain has disclosed in the tenuigenin that RASGEF1A albumen is positioned transfectional cell (Fig. 2 A).

3.RASGEF1A relation with the growth of ICC cell

Carry out the colony forming assay method to check the carcinogenic activity of RASGEF1A in the COS7 cell.The result is, compare with the transfection of using control plasmid pCAGGS-n3Fc-Mock or pCAGGS-n3Fc-RASGEF1A (-), viewed colony digital display work increases (Fig. 3 A and 3B) when cell is used plasmid (pCAGGS-n3Fc-RASGEF1A) transfection of expressing RASGEF1A.For further assessing its latent effect in the cell growth, the plasmid psiH1BX-RASGEF1A-siB and the psiH1BX-RASGEF1A-siE of RASGEF1A specific siRNA and neomycin resistance gene expressed in preparation.With arbitrary above-mentioned plasmid or with control plasmid (psiH1BX-siEGFP, psiH1BX-Mock) transfection SSP25ICC cell.Sxemiquantitative RT-PCR has shown with the transfection of control plasmid and has compared that RASGEF1A expresses the inhibition (Fig. 3 C) that is subjected to psiRNA-BX-RASGEF1A-siB and psiH1BX-RASGEF1A-siE transfection.Particularly, and compare with the control plasmid cells transfected, the transfectional cell that suppresses with siRNA-B and siRNA-E has obvious growth sluggishness (Fig. 3 D).The expression that these data show RASGEF1A is for the growth of ICC cell and/or survive most important.

4.RASGEF1A the Ras Nucleotide activity of dissociating

Because RASEGF1A contains the RasGEF structural domain of inferring, therefore study its GDP activity of dissociating.Add subsequently and contain or do not contain the proteic reaction mixture of reorganization RASGEF1A, and be combined with the RAS of GDP with the scintillometer measurement.SOS (a kind of known RASGEF albumen) is as positive control.SOS reduces GDP mating type K-RAS about 40%.Similarly, RASGEF1A reduces about 50% (Fig. 4 A, the upper left quarter) of GDP mating type K-RAS.Its GDP that is bonded to H-RAS and N-RAS (Fig. 4 A, upper right quarter and bottom) of about 40% and 80% that also dissociated respectively.These data show that RASEGF1A demonstrates the GDP activity of dissociating for all three kinds checked RAS albumen.

5.RASGEF1A activation to RAS

Because RASGEF1A contains the RASGEF structural domain of inferring, therefore further whether research RASGEF1A has the activity that stimulates RAS.Use COS7 cell and the control cells of expressing external source RASGEF1A to measure this activity.The immunoblotting assay that uses anti-FLAG antibody that the extract with the cell of pxFLAG-RASGEF1A plasmid transfection is carried out has confirmed that the external source RASGEF1A in the transfectional cell expresses (Fig. 4 B, upper left quarter).Recombinate " drop-down " assay method of Raf-1 (effector of a kind of RAS) of pair cell extract or control cells extract.The result is, expresses corresponding toly being to use anti-general Ras antibody to increase (Fig. 4 B, upper right quarter) by the immunoblotting assay amount with the interactional Ras of RBD Raf-1 that measure with RASGEF1A, shows that RASGEF1A has strengthened the activity of RAS.

6.RASGEF1A effect to the cancer cells migration

Reported that the RAS family protein plays an important role in cell migration and cell growth.Therefore, the relation of migration of research RASGEF1A and cancer cells and intrusion.Set up COS7-RASGEF1A cell and the control cells (COS7-Mock) of expressing external source RASGEF1A and be used for the wound healing assay method.Shown in Fig. 5 A, COS7-RASGEF1A cell fast transferring also is full of wound, and obviously faster than the COS7-Mock cell, hint RASGEF1A relates to cell migration.In addition, be the effect of investigation RASGEF1A, use Matrigel (transwell) assay method of boring a hole invading.Consistent with the result of wound healing assay method, comparing the number that passes the COS7-RASGEF1A cell of invading the chamber hole with the COS7-Mock cell increases (Fig. 5 B).Because these two kinds of cells have similar multiplication rate, so these data declarations RASGEF1A relates to cell migration.

III. discussion of results

The inventor has proved expression that RASGEF1A raises and to the guanylic acid exchange activity of K-RAS, H-RAS and N-RAS at this in people ICC, hint in the oncogenesis that is expressed in ICC of its rising to play considerable effect.Up to now, reported about 20 members that belong to Ras-GEF family, they enjoy core catalytic domain (the Boguski MS ﹠amp with 5 structure conserved regions; McCornickF, Nature 1993,366:643-54; Rebhun JF et al., J Biol Chem 2000,275:13406-10).As other Ras-GEF member, RASGEF1A contains this 5 conserved regions.In addition, it has REM (Ras exchanges motif) or RasGEFN structural domain between codon 49 and 178, be considered to stablize the major spiral hairpin structure of opening the GTP binding pocket.Yet, because RASGEF1A lacks any other conservative territory, such as Db1 homology (DH) territory, pleckstrin (pleckstrin) homology (PH) territory, EF hand, be rich in the C1 territory and the pdz domain of halfcystine, its may bring into play with other member such as SOS, GRF1, RasGRP or PDZ-GEF different function (Quilliam LA et al., Progress inNucleic Acid Research and Molecular Biology 2002,71:391-444).In the present invention, disclose RASGEF1A and had guanylic acid exchange activity to K-RAS, H-RAS and N-RAS.Other Ras-GEF member has different substrate spectrums; For example, SOS1 regulates K-RAS, H-RAS, N-RAS, R-RAS2, R-RAS3 and Rac1, but does not regulate R-RAS; Ras-GRP2 stimulates K-RAS, H-RAS, N-RAS, R-RAS, R-RAS2, Rap1A, Rap2A, but do not stimulate H-RAS (MitinN et al., Current Biology 2005,15:R563-74).Therefore, RASGEF1A also may not have the substrate of investigation in specificity activation the present invention research, and may relate to crosstalk (crosstalk) of Ras-signal transduction path.

Recently studies show that the conduction of Ras signal is not only relevant, also relevant with cell movement and cell adhesion with survival with cell proliferation.Activated Ras and multiple downstream effect thing interact and regulate them, and described downstream effect thing stimulates the different signal transduction path that comprises Raf/MEK/ERK and phosphoinositide 3 kinases (PI3K)/Akt, RalGDS/Ral and Tiam1/Rac1 approach.Ji Lei evidence shows that Raf/MEK/ERK is relevant with the cell proliferation of Ras mediation day by day.Raf binding assay proof activated RAS when RASGEF1A exists increases, and therefore thinks and expressing the cell middle and lower reaches Raf/MEK/ERK enhancing of RASGEF1A.Corresponding toly be, suppress RASGEF1A by importing specific siRNA, reduced the growth of ICC cell, this conducts the viewpoint of giving the growth of cancer cells facilitation effect with enhanced RAF/MEK/ERK signal is consistent.Using COS7 and NIH3T3 inoblast to test in the colony forming assay method of RASGEF1A carcinogenic activity, colony number does not change between cell of expressing external source RASGEF1A and contrast.Therefore, import RASGEF1A probably separately and be not enough to cause COS7 or the fibroblastic conversion of NIH3T3.Yet the carcinogenic activity of RASGEF1A is considered to tissue and/or cell type is dependent, and the siRNA test shows that RASGEF1A is absolutely necessary to cell growth or the propagation of keeping ICC.Therefore, the inhibition to its guanylic acid exchange activity may become ICC treatment selection likely.Outside its growth-promoting effect, reported that Ras plays a role in cell movement by different mechanisms.The breast cancer cell that enhanced PI3 kinase activity causes increasing is invaded, and it mediates by beta 4 integrin in Rac1 dependency mode.PI3K also activates RacGEF, and induces Actin muscle reorganization and film wrinkling, and they are relevant with cell movement.In addition, Ras has strengthened Tiam1, the activated form Rac1 that generation is relevant with migration and Actin muscle-cytoskeleton.In inoblast, the activation of H-RAS has suppressed activity (Paul EH et al., Mol Biol Cell 2002, the 13:2256-65 of integrin (cell surface receptor that plays an important role) in extracellular matrix adheres to; SechlerJL et al., J Cell Sci 2000,113:1491-8).According to the present invention, find that RASGEF1A activates RAS albumen, and the heterogenous expression of RASGEF1A strengthens cell movement in the Matrigel assay method.Although require further study the mechanism that increases mobility by RASGEF1A of illustrating, propose RASGEF1A and strengthen cell movement by activating PI3K or Tiam1.

The inventor has further studied the relation between K-Ras mutation status and the RASGEF1A expression level, but does not find to exist between them any relation (data not shown).Because outside K-Ras, the RASGEF1A of rising expresses and has also activated H-Ras and N-Ras, therefore compares with the sudden change among the K-Ras, and the accumulation of RASGEF1A may have other effect in the oncogenesis of ICC.Owing to the inhibition that causes by siRNA inhibition RASGEF1A cancer cells, so RASGEF1A may be a kind of ICC treatment target likely.

Industrial applicibility

The cancer gene expression analysis that utilizes the laser capture dissection and the combination of genome range cDNA microarray to carry out described here has identified the specific gene that is used for cancer prevention and treatment as target.According to the expression of these difference expression gene subclass, the invention provides the molecular diagnostic markers that is used to identify and detect cancer.

Methods described herein also can be used for identifying other molecule target of prevention, diagnosis and treatment cancer.The data that this paper provided have increased the overall understanding to cancer, have promoted the exploitation of new diagnosis policy, and the clue of identifying the molecule target of curative drug and preventative medicament is provided.These information help more deep understanding tumour to take place, and provide exploitation to be used to diagnose, treat and the index of the New Policy of final preventing cancer.

All patents, patent application and publication that complete income is quoted herein are as a reference.

In addition, although the present invention describes in detail with reference to specific embodiments, should understand, above describing is exemplary and indicative in essence, intention illustration the present invention and preferred embodiment thereof.By normal experiment, those skilled in the art will be easy to recognize and can carry out multiple change and modification to the present invention under the prerequisite that does not deviate from the spirit and scope of the present invention.So, the invention is intended to limit, but not above describe with claims and equivalent thereof.

Sequence table

＜110〉Oncotherapy Science Inc (ONCOTHERAPY SCIENCE, INC.)

＜120〉cancer related gene RASGEF 1 A

<130>ONC-A0513P

<150>US60/704,054

<151>2005-07-28

<160>20

<170>PatentIn?version?3.3

<210>1

<211>3266

<212>DNA

＜213〉human (Homo sapiens)

<220>

<221>CDS

<222>(86)..(1555)

<400>1

ggtgcttgct?tggctcgcgg?gcagaggagc?cgccgctcgc?tggacgccgg?accgggcagg 60

acggcgcggg?gagccggcgg?ccaga?atg?ttc?ctg?gag?ccc?cag?gaa?act?atg 112

Met?Phe?Leu?Glu?Pro?Gln?Glu?Thr?Met

1 5

ccc?cag?acg?tcc?gtt?gtc?ttc?tcc?agc?atc?ctt?ggg?ccc?agc?tgt?agc 160

Pro?Gln?Thr?Ser?Val?Val?Phe?Ser?Ser?Ile?Leu?Gly?Pro?Ser?Cys?Ser

10 15 20 25

gga?cag?gtg?cag?cct?ggc?atg?ggg?gag?cgt?gga?ggc?ggg?gcc?ggt?ggc 208

Gly?Gln?Val?Gln?Pro?Gly?Met?Gly?Glu?Arg?Gly?Gly?Gly?Ala?Gly?Gly

30 35 40

ggc?tcc?ggg?gac?ctc?atc?ttc?caa?gat?gga?cac?ctc?atc?tct?ggg?tcc 256

Gly?Ser?Gly?Asp?Leu?Ile?Phe?Gln?Asp?Gly?His?Leu?Ile?Ser?Gly?Ser

45 50 55

ctg?gag?gcc?ctg?atg?gag?cac?ctt?gtt?ccc?acg?gtg?gac?tat?tac?ccc 304

Leu?Glu?Ala?Leu?Met?Glu?His?Leu?Val?Pro?Thr?Val?Asp?Tyr?Tyr?Pro

60 65 70

gat?agg?acg?tac?atc?ttc?acc?ttt?ctc?ctg?agc?tcc?cgg?gtc?ttt?atg 352

Asp?Arg?Thr?Tyr?Ile?Phe?Thr?Phe?Leu?Leu?Ser?Ser?Arg?Val?Phe?Met

75 80 85

ccc?cct?cat?gac?ctg?ctg?gcc?cgc?gtg?ggg?cag?atc?tgc?gtg?gag?cag 400

Pro?Pro?His?Asp?Leu?Leu?Ala?Arg?Val?Gly?Gln?Ile?Cys?Val?Glu?Gln

90 95 100 105

aag?cag?cag?ctg?gaa?gcc?ggg?cct?gaa?aag?gcc?aag?ctg?aag?tct?ttc 448

Lys?Gln?Gln?Leu?Glu?Ala?Gly?Pro?Glu?Lys?Ala?Lys?Leu?Lys?Ser?Phe

110 115 120

tca?gcc?aag?atc?gtg?cag?ctc?ctg?aag?gag?tgg?acc?gag?gcc?ttc?ccc 496

Ser?Ala?Lys?Ile?Val?Gln?Leu?Leu?Lys?Glu?Trp?Thr?Glu?Ala?Phe?Pro

125 130 135

tat?gac?ttc?cag?gat?gag?aag?gcc?atg?gcc?gag?ctg?aaa?gcc?atc?aca 544

Tyr?Asp?Phe?Gln?Asp?Glu?Lys?Ala?Met?Ala?Glu?Leu?Lys?Ala?Ile?Thr

140 145 150

cac?cgt?gtc?acc?cag?tgt?gat?gag?gag?aat?ggc?aca?gtg?aag?aag?gcc 592

His?Arg?Val?Thr?Gln?Cys?Asp?Glu?Glu?Asn?Gly?Thr?Val?Lys?Lys?Ala

155 160 165

att?gcc?cag?atg?aca?cag?agc?ctg?ttg?ctg?tcc?ttg?gct?gcc?cgg?agc 640

Ile?Ala?Gln?Met?Thr?Gln?Ser?Leu?Leu?Leu?Ser?Leu?Ala?Ala?Arg?Ser

170 175 180 185

cag?ctc?cag?gaa?ctg?cga?gag?aag?ctc?cgg?cca?ccg?gct?gta?gac?aag 688

Gln?Leu?Gln?Glu?Leu?Arg?Glu?Lys?Leu?Arg?Pro?Pro?Ala?Val?Asp?Lys

190 195 200

ggg?ccc?atc?ctc?aag?acc?aag?cca?cca?gcc?gcc?cag?aag?gac?atc?ctg 736

Gly?Pro?Ile?Leu?Lys?Thr?Lys?Pro?Pro?Ala?Ala?Gln?Lys?Asp?Ile?Leu

205 210 215

ggc?gtg?tgc?tgc?gac?ccc?ctg?gtg?ctg?gcc?cag?cag?ctg?act?cac?att 784

Gly?Val?Cys?Cys?Asp?Pro?Leu?Val?Leu?Ala?Gln?Gln?Leu?Thr?His?Ile

220 225 230

gag?ctg?gac?agg?gtc?agc?agc?att?tac?cct?gag?gac?ttg?atg?cag?atc 832

Glu?Leu?Asp?Arg?Val?Ser?Ser?Ile?Tyr?Pro?Glu?Asp?Leu?Met?Gln?Ile

235 240 245

gtc?agc?cac?atg?gac?tcc?ttg?gac?aac?cac?agg?tgc?cga?ggg?gac?ctg 880

Val?Ser?His?Met?Asp?Ser?Leu?Asp?Asn?His?Arg?Cys?Arg?Gly?Asp?Leu

250 255 260 265

acc?aag?acc?tac?agc?ctg?gag?gcc?tat?gac?aac?tgg?ttc?aac?tgc?ctg 928

Thr?Lys?Thr?Tyr?Ser?Leu?Glu?Ala?Tyr?Asp?Asn?Trp?Phe?Asn?Cys?Leu

270 275 280

agc?atg?ctg?gtg?gcc?act?gag?gtg?tgc?cgg?gtg?gtg?aag?aag?aaa?cac 976

Ser?Met?Leu?Val?Ala?Thr?Glu?Val?Cys?Arg?Val?Val?Lys?Lys?Lys?His

285 290 295

cgg?acc?cgc?atg?ttg?gag?ttc?ttc?att?gat?gtg?gcc?cgg?gag?tgc?ttc 1024

Arg?Thr?Arg?Met?Leu?Glu?Phe?Phe?Ile?Asp?Val?Ala?Arg?Glu?Cys?Phe

300 305 310

aac?atc?ggg?aac?ttc?aac?tcc?atg?atg?gcc?atc?atc?tct?ggc?atg?aac 1072

Asn?Ile?Gly?Asn?Phe?Asn?Ser?Met?Met?Ala?Ile?Ile?Ser?Gly?Met?Asn

315 320 325

ctc?agt?cct?gtg?gca?agg?ctg?aag?aaa?act?tgg?tcc?aag?gtc?aag?aca 1120

Leu?Ser?Pro?Val?Ala?Arg?Leu?Lys?Lys?Thr?Trp?Ser?Lys?Val?Lys?Thr

330 335 340 345

gcc?aag?ttt?gat?gtc?ttg?gag?cat?cac?atg?gac?ccg?tcc?agc?aac?ttc 1168

Ala?Lys?Phe?Asp?Val?Leu?Glu?His?His?Met?Asp?Pro?Ser?Ser?Asn?Phe

350 355 360

tgc?aac?tac?cgt?aca?gcc?ctg?cag?ggg?gcc?acg?cag?agg?tcc?cag?atg 1216

Cys?Asn?Tyr?Arg?Thr?Ala?Leu?Gln?Gly?Ala?Thr?Gln?Arg?Ser?Gln?Met

365 370 375

gcc?aac?agc?agc?cgt?gaa?aag?atc?gtc?atc?cct?gtg?ttc?aac?ctc?ttc 1264

Ala?Asn?Ser?Ser?Arg?Glu?Lys?Ile?Val?Ile?Pro?Val?Phe?Asn?Leu?Phe

380 385 390

gtt?aag?gac?atc?tac?ttc?ctg?cac?aaa?atc?cat?acc?aac?cac?ctg?ccc 1312

Val?Lys?Asp?Ile?Tyr?Phe?Leu?His?Lys?Ile?His?Thr?Asn?His?Leu?Pro

395 400 405

aac?ggg?cac?att?aac?ttt?aag?aaa?ttc?tgg?gag?atc?tcc?aga?cag?atc 1360

Asn?Gly?His?Ile?Asn?Phe?Lys?Lys?Phe?Trp?Glu?Ile?Ser?Arg?Gln?Ile

410 415 420 425

cat?gag?ttc?atg?aca?tgg?aca?cag?gta?gag?tgt?cct?ttc?gag?aag?gac 1408

His?Glu?Phe?Met?Thr?Trp?Thr?Gln?Val?Glu?Cys?Pro?Phe?Glu?Lys?Asp

430 435 440

aag?aag?att?cag?agt?tac?ctg?ctc?acg?gcg?ccc?atc?tac?agc?gag?gaa 1456

Lys?Lys?Ile?Gln?Ser?Tyr?Leu?Leu?Thr?Ala?Pro?Ile?Tyr?Ser?Glu?Glu

445 450 455

gct?ctc?ttc?gtc?gcc?tcc?ttt?gaa?agt?gag?ggt?ccc?gag?aac?cac?atg 1504

Ala?Leu?Phe?Val?Ala?Ser?Phe?Glu?Ser?Glu?Gly?Pro?Glu?Asn?His?Met

460 465 470

gaa?aaa?gac?agc?tgg?aag?acc?ctc?agg?acc?acc?ctt?ctg?aac?aga?gcc 1552

Glu?Lys?Asp?Ser?Trp?Lys?Thr?Leu?Arg?Thr?Thr?Leu?Leu?Asn?Arg?Ala

475 480 485

tga?ggcggatgca?gcccgcgacg?ccagaggaag?cacgtgcact?aactgggttt 1605

aaattttgac?tgatgtgggt?tgagatgagg?aggcctcact?ggttggggtc?cattttgtat 1665

ataactttta?tgagaaaaaa?atggtaatta?tttcacgcat?caacctttgg?cacttacaaa 1725

gttttttttg?tttattttaa?ataacagggc?agggccctgc?tttggggagg?gggaggggag 1785

agtatcatgg?gagatggtat?ccatgataac?atcttattct?aatgaaatgt?agatttttat 1845

tttctacttt?tgattattaa?catcttatga?aaaaaatatt?ttaaaaaacc?cagccaaaac 1905

caacgtgagc?cctgcctgct?cggacgcctt?tccagccagt?gtctctgacg?tcggggttag 1965

tgccttagag?ggtactgggg?tctggtcttc?ctgctctgtg?gtttgggctg?cggtgagtcc 2025

cactccacct?gggcgcctgc?cctcaggagc?ctgggctgcg?aggctccata?ggagggctgg 2085

tggctgggag?gtcgcgtccg?cacacttctg?gaagtgagcc?tttgagtacg?ggctgtccaa 2145

agtttacatt?ttcattttcc?tttcagggat?ttgtggggtc?agggaggggc?agggggcacc 2205

tggcagcata?ttttctgtga?caatgtgtcc?agcaaatcat?tcttcaacta?cattttagaa 2265

aggaggaaat?ctaaaataag?gtaagggagg?gaagcatgga?gttgtcagtt?ttctgggctg 2325

tgactgaaag?acacactgag?ctgtgatgaa?gaaaaataca?tggccgactc?cagggtggtg 2385

acatttagag?ctagtcttga?aacctatcat?ctacagaggg?gagggcagcc?aacagccctc 2445

ttcccacctg?ggtaggcagc?gccctaattg?gaattggaaa?cagaaaattc?gccaggccat 2505

actgctggag?cccattcaga?taaaactgcc?caatactgag?aggtgttttc?tacacccagc 2565

tagaggagca?cactccattt?tcccatgtct?gacttcgtgg?tgtgagccct?gggccctact 2625

gaccatggcg?caggacagct?gtccttcaga?aagcacacgg?tcaatccacg?tggaccgtct 2685

ccctcgcagg?aactccgcat?ccttgtccct?ctctgcattc?ccagtttccg?caggagcctt 2745

gatcaatggg?gaagcctggg?tgaggatggg?ccaggtccca?attcccaaag?ctcctggaag 2805

agcctgaaga?cattgggaaa?ggctgggcct?ggggaggagg?cagccctggg?cccgctgccc 2865

atgcctctgg?tcctgggtgg?agcaggaata?gttccactgt?attgtcacag?tgtgtttgca 2925

ctttctgagg?ttctagctag?tacagattgt?atattgatag?tacatattgc?tttgtttatg 2985

tctttgagat?gagaaaggct?taaaacttga?gaatatatat?ttggaataca?gccttagaac 3045

ggtttctgta?cacatccacg?tgcacttcac?gggtgatcag?ttctagtacc?tacttgaaac 3105

agtgtctgtc?tgctacttta?ttttcccaat?ttgatacata?ccctgatttg?atgttttggt 3165

atttgagatg?aactctgagt?atgaagctgt?accataatgc?aggacgtcag?ttttggtgtg 3225

actggacata?cttgcttcaa?taaaagaata?catcactcccc 3266

<210>2

<211>489

<212>PRT

＜213〉human (Homo sapiens)

<400>2

Met?Phe?Leu?Glu?Pro?Gln?Glu?Thr?Met?Pro?Gln?Thr?Ser?Val?Val?Phe

1 5 10 15

Ser?Ser?Ile?Leu?Gly?Pro?Ser?Cys?Ser?Gly?Gln?Val?Gln?Pro?Gly?Met

20 25 30

Gly?Glu?Arg?Gly?Gly?Gly?Ala?Gly?Gly?Gly?Ser?Gly?Asp?Leu?Ile?Phe

35 40 45

Gln?Asp?Gly?His?Leu?Ile?Ser?Gly?Ser?Leu?Glu?Ala?Leu?Met?Glu?His

50 55 60

Leu?Val?Pro?Thr?Val?Asp?Tyr?Tyr?Pro?Asp?Arg?Thr?Tyr?Ile?Phe?Thr

65 70 75 80

Phe?Leu?Leu?Ser?Ser?Arg?Val?Phe?Met?Pro?Pro?His?Asp?Leu?Leu?Ala

85 90 95

Arg?Val?Gly?Gln?Ile?Cys?Val?Glu?Gln?Lys?Gln?Gln?Leu?Glu?Ala?Gly

100 105 110

Pro?Glu?Lys?Ala?Lys?Leu?Lys?Ser?Phe?Ser?Ala?Lys?Ile?Val?Gln?Leu

115 120 125

Leu?Lys?Glu?Trp?Thr?Glu?Ala?Phe?Pro?Tyr?Asp?Phe?Gln?Asp?Glu?Lys

130 135 140

Ala?Met?Ala?Glu?Leu?Lys?Ala?Ile?Thr?His?Arg?Val?Thr?Gln?Cys?Asp

145 150 155 160

Glu?Glu?Asn?Gly?Thr?Val?Lys?Lys?Ala?Ile?Ala?Gln?Met?Thr?Gln?Ser

165 170 175

Leu?Leu?Leu?Ser?Leu?Ala?Ala?Arg?Ser?Gln?Leu?Gln?Glu?Leu?Arg?Glu

180 185 190

Lys?Leu?Arg?Pro?Pro?Ala?Val?Asp?Lys?Gly?Pro?Ile?Leu?Lys?Thr?Lys

195 200 205

Pro?Pro?Ala?Ala?Gln?Lys?Asp?Ile?Leu?Gly?Val?Cys?Cys?Asp?Pro?Leu

210 215 220

Val?Leu?Ala?Gln?Gln?Leu?Thr?His?Ile?Glu?Leu?Asp?Arg?Val?Ser?Ser

225 230 235 240

Ile?Tyr?Pro?Glu?Asp?Leu?Met?Gln?Ile?Val?Ser?His?Met?Asp?Ser?Leu

245 250 255

Asp?Asn?His?Arg?Cys?Arg?Gly?Asp?Leu?Thr?Lys?Thr?Tyr?Ser?Leu?Glu

260 265 270

Ala?Tyr?Asp?Asn?Trp?Phe?Asn?Cys?Leu?Ser?Met?Leu?Val?Ala?Thr?Glu

275 280 285

Val?Cys?Arg?Val?Val?Lys?Lys?Lys?His?Arg?Thr?Arg?Met?Leu?Glu?Phe

290 295 300

Phe?Ile?Asp?Val?Ala?Arg?Glu?Cys?Phe?Asn?Ile?Gly?Asn?Phe?Asn?Ser

305 310 315 320

Met?Met?Ala?Ile?Ile?Ser?Gly?Met?Asn?Leu?Ser?Pro?Val?Ala?Arg?Leu

325 330 335

Lys?Lys?Thr?Trp?Ser?Lys?Val?Lys?Thr?Ala?Lys?Phe?Asp?Val?Leu?Glu

340 345 350

His?His?Met?Asp?Pro?Ser?Ser?Asn?Phe?Cys?Asn?Tyr?Arg?Thr?Ala?Leu

355 360 365

Gln?Gly?Ala?Thr?Gln?Arg?Ser?Gln?Met?Ala?Asn?Ser?Ser?Arg?Glu?Lys

370 375 380

Ile?Val?Ile?Pro?Val?Phe?Asn?Leu?Phe?Val?Lys?Asp?Ile?Tyr?Phe?Leu

385 390 395 400

His?Lys?Ile?His?Thr?Asn?His?Leu?Pro?Asn?Gly?His?Ile?Asn?Phe?Lys

405 410 415

Lys?Phe?Trp?Glu?Ile?Ser?Arg?Gln?Ile?His?Glu?Phe?Met?Thr?Trp?Thr

420 425 430

Gln?Val?Glu?Cys?Pro?Phe?Glu?Lys?Asp?Lys?Lys?Ile?Gln?Ser?Tyr?Leu

435 440 445

Leu?Thr?Ala?Pro?Ile?Tyr?Ser?Glu?Glu?Ala?Leu?Phe?Val?Ala?Ser?Phe

450 455 460

Glu?Ser?Glu?Gly?Pro?Glu?Asn?His?Met?Glu?Lys?Asp?Ser?Trp?Lys?Thr

465 470 475 480

Leu?Arg?Thr?Thr?Leu?Leu?Asn?Arg?Ala

485

<210>3

<211>19

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the synthetic primer of RT-PCR

<400>3

caagatgaga?ttggcatgg 19

<210>4

<211>19

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the synthetic primer of RT-PCR

<400>4

tctccttaga?gagaagtgg 19

<210>5

<211>23

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the synthetic primer of RT-PCR

<400>5

tttgcacttt?ctgaggttct?agc 23

<210>6

<211>23

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the synthetic primer of RT-PCR

<400>6

gtcctgcatt?atggtacagc?ttc 23

<210>7

<211>30

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the synthetic primer of RT-PCR

<400>7

attgaattcc?ggccagaatg?ttcctggagc 30

<210>8

<211>31

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the synthetic primer of RT-PCR

<400>8

aatctcgagg?gctctgttca?gaagggtggt?c 31

<210>9

<211>19

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the synthetic target sequence of siRNA

<400>9

gagaatggca?cagtgaaga 19

<210>10

<211>51

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial synthetic oligonucleotide of siRNA

<400>10

tcccgagaat?ggcacagtga?agattcaaga?gatcttGaGt?gtgccattct?c 51

<210>11

<211>51

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial synthetic oligonucleotide of siRNA

<400>11

aaaagagaat?ggcacagtga?agatctcttg?aatcttcact?gtgccattct?c 51

<210>12

<211>47

<212>DNA

＜213〉artificial

<220>

＜223〉siRNA hair clip design

<400>12

gagaatggca?cagtgaagat?tcaagagatc?ttcactgtgc?cattctc 47

<210>13

<211>19

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the synthetic target sequence of siRNA

<400>13

catctacttc?ctgcacaaa 19

<210>14

<211>51

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial synthetic oligonucleotide of siRNA

<400>14

tccccatcta?cttcctgcac?aaattcaaga?gatttgtgca?ggaagtagat?g 51

<210>15

<211>51

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial synthetic oligonucleotide of siRNA

<400>15

aaaacatcta?cttcctgcac?aaatctcttg?aatttgtgca?ggaagtagat?g 51

<210>16

<211>47

<212>DNA

＜213〉artificial

<220>

＜223〉siRNA hair clip design

<400>16

catctacttc?ctgcacaaat?tcaagagatt?tgtgcaggaa?gtagatg 47

<210>17

<211>19

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the synthetic target sequence of siRNA

<400>17

gaagcagcac?gacttcttc 19

<210>18

<211>51

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial synthetic oligonucleotide of siRNA

<400>18

tcccgaagca?gcacgacttc?ttcttcaaga?gagaagaagt?cgtgctgctt?c 51

<210>19

<211>51

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the artificial synthetic oligonucleotide of siRNA

<400>19

aaaagaagca?gcacgacttc?ttctctcttg?aagaagaagt?cgtgctgctt?c 51

<210>20

<211>47

<212>DNA

＜213〉artificial

<220>

＜223〉siRNA hair clip design

<400>20

gaagcagcac?gacttcttct?tcaagagaga?agaagtcgtg?ctgcttc 47

Claims (28)

1. one kind is used for diagnosing experimenter's the cancer or the tendentious method of generation cancer, it may further comprise the steps: measure RASGEF1A expression of gene level in deriving from experimenter's biological sample, the rising that wherein said expression level is compared with described gene normal control level shows that described experimenter suffers from cancer or risky generation cancer.

2. the process of claim 1 wherein that described expression level is than normal control level height at least 10%.

3. the process of claim 1 wherein and measure described expression level by any method that is selected from down group:

(a) mRNA of detection RASGEF1A gene;

(b) protein of detection RASGEF1A genes encoding; With

(c) the proteinic biologic activity of detection RASGEF1A genes encoding.

4. the process of claim 1 wherein that the described experimenter's of deriving from biological sample comprises epithelial cell.

5. the process of claim 1 wherein that the described experimenter's of deriving from biological sample comprises cancer cells.

6. the process of claim 1 wherein that the described experimenter's of deriving from biological sample comprises carcinous epithelial cell.

7. the process of claim 1 wherein that described cancer is selected from ICC, bladder cancer, lung cancer, cancer of the stomach, colorectal carcinoma, prostate cancer, chronic myeloid leukemia, kidney, soft tissue sarcoma, carcinoma of the pancreas and spermocytoma.

8. test kit, its comprise can with RASGEF1A gene transcription or translation product bonded detection reagent.

9. a screening is used for the treatment of or the method for the medicament of preventing cancer, and it may further comprise the steps:

A) contacted with RASGEF1A polypeptide or its fragment by the reagent agent;

B) detect described polypeptide and be subjected to combining between the reagent agent with described; And

C) select to be subjected to the reagent agent with described polypeptide bonded.

10. a screening is used for the treatment of or the method for the medicament of preventing cancer, and it may further comprise the steps:

A) contacted with RASGEF1A polypeptide or its fragment by the reagent agent;

B) biologic activity of the described polypeptide of detection; And

C) select with do not have the condition that is subjected to the reagent agent under detected biologic activity compare the biologic activity that suppresses described polypeptide be subjected to the reagent agent.

11. a screening is used for the treatment of or the method for the medicament of preventing cancer, it may further comprise the steps:

(a) be subjected under the condition of reagent agent in existence, RASGEF1A polypeptide or its fragment are contacted with RAS;

(b) detect the RAS activity; And

(c) select with do not have the condition that is subjected to the reagent agent under detected RAS activity compare and reduce that RAS is active to be subjected to the reagent agent.

12. a screening is used for the treatment of or the method for the medicament of preventing cancer, it may further comprise the steps:

A) will be subjected to reagent agent and the cells contacting of expressing the RASGEF1A gene;

B) detect RASGEF1A expression of gene level; And

C) select with do not have the condition that is subjected to the reagent agent under detected expression level compare reduce described expression of gene level be subjected to the reagent agent.

13. the method for claim 12, wherein said cell derives from ICC, bladder cancer, lung cancer, cancer of the stomach, colorectal carcinoma, prostate cancer, chronic myeloid leukemia, kidney, soft tissue sarcoma, carcinoma of the pancreas or spermocytoma.

14. a screening is used for the treatment of or the method for the medicament of preventing cancer, it may further comprise the steps:

A) will be subjected to reagent agent and the cells contacting that has wherein imported carrier, the reporter gene that described carrier comprises RASGEF1A gene transcription regulatory region and expresses under this transcriptional regulatory district control;

B) expression or the activity of the described reporter gene of measurement; And

C) select with do not have the condition that is subjected to the reagent agent under detected expression or actively compare the expression that reduces described reporter gene or actively be subjected to the reagent agent.

15. each method of claim 9-14, wherein said cancer is selected from ICC, bladder cancer, lung cancer, cancer of the stomach, colorectal carcinoma, prostate cancer, chronic myeloid leukemia, kidney, soft tissue sarcoma, carcinoma of the pancreas and spermocytoma.

16. one kind is used for the treatment of or the composition of preventing cancer, it comprises medicament and the pharmaceutically acceptable carrier that each method of claim 9-15 is selected that pass through as the pharmaceutically significant quantity of activeconstituents.

17. one kind is used for the treatment of or the composition of preventing cancer, it comprises the antisense polynucleotides or the siRNA at the polynucleotide of RASGEF1A genes encoding of significant quantity pharmaceutically.

18. the composition of claim 17, wherein said siRNA comprise the sense strand of the RASGEF1A gene of the nucleotide sequence that contains SEQ ID NO:9 or 13.

19. the composition of claim 18, wherein said siRNA has general formula 5 '-[A]-[B]-[A ']-3 ', wherein [A] is the ribonucleoside acid sequence corresponding to the sequence of SEQ ID NO:9 or 13, the ribonucleotide ring sequence that [B] is made up of 3-23 Nucleotide, and [A '] be and [A] complementary ribonucleoside acid sequence.

20. one kind is used for the treatment of or the composition of preventing cancer, its comprise significant quantity pharmaceutically can with RASGEF1A polypeptide bonded antibody or its fragment.

21. each composition of claim 16-20, wherein said cancer is selected from ICC, bladder cancer, lung cancer, cancer of the stomach, colorectal carcinoma, prostate cancer, chronic myeloid leukemia, kidney, soft tissue sarcoma, carcinoma of the pancreas and spermocytoma.

22. one kind is used for the treatment of or prevents method for cancer among the experimenter, it comprises the step of the experimenter being used the medicament that obtains by each method of claim 9-15.

23. one kind is used for the treatment of or prevents method for cancer among the experimenter, it comprises uses each the step of composition of claim 16-20 to the experimenter.

24. one kind is used for the treatment of or prevents method for cancer among the experimenter, it comprise to the experimenter use significant quantity pharmaceutically can with RASGEF1A polypeptide bonded antibody or the segmental step of its immunologic competence.

25. one kind is used for the treatment of or prevents method for cancer among the experimenter, it comprises the step of the experimenter being used vaccine, described vaccine comprises (a) RASGEF1A polypeptide, (b) the immunologic competence fragment of described polypeptide, or (c) coding said polypeptide or segmental polynucleotide.

26. a method that is used for inducing antitumor immunity power, it comprises APC and RASGEF1A polypeptide or its fragment, coding said polypeptide or segmental polynucleotide or comprises the step that the carrier of described polynucleotide contacts.

27. the method that is used for inducing antitumor immunity power of claim 26, it further comprises the step of the experimenter being used APC.

28. each method of claim 22-27, wherein said cancer is selected from ICC, bladder cancer, lung cancer, cancer of the stomach, colorectal carcinoma, prostate cancer, chronic myeloid leukemia, kidney, soft tissue sarcoma, carcinoma of the pancreas and spermocytoma.

Images