CN102274500A - Medicament for treating or preventing cancers - Google Patents

Abstract

The invention relates to a medicament for treating or preventing cancers. The medicament contains effective dose of human transcription positive and auxiliary factor 4 (PC4) antagonists, wherein in particular, the PC4 antagonists are antibodies against the PC4; and the antibodies are subtype specific antibodies against the PC4 and are chimeric antibodies. The medicament achieves the aim of treating or preventing cancers by inhibiting the expression level or function of the PC4.

Description

A kind of being used for the treatment of or the medicine of prophylaxis of cancer

The present invention be that JIUYUE in 2008 7 days, application number are 200810160891.1 the applying date, name is called the dividing an application of Chinese invention patent application of " method and the medicine of treatment or prophylaxis of cancer ".

Technical field

The present invention relates to treat or method and the medicine and the method for screening anticancer medicine of prophylaxis of cancer.

Background technology

It is a kind of single-stranded DNA binding protein that the people transcribes positive cofactor 4 (PC4 also claims the homology analog of p14, p15, Sub1 etc.), and this albumen is made up of 127 amino acid residues and near the N-end several serine enrichment regions is arranged.PC4 cloned and be accredited as a kind of can by with many sequence-specifics and cell-specific regulon directly be used for mediating the activatory general positive cofactor of many gene transcription (referring to document Ge etc., Cell (1994) 78:513-523; Kretzschmar, M. etc., Cell (1994) 78:525-534).Direct acting these regulon of PC4 comprise necessary important factor in the pathological process of the generating process of many nuclear hormone receptors, tumor suppression, cancer protein and tumor and other human diseasess.

Tumor suppressor protein P53 directly and PC4 albumen interact at transcriptional level, thereby controlled the expression of PC4.In addition, PC4 activates as P53 unique that son can be regulated many participation cell cycles, apoptosis, DNA repairs and the gene transcription of other cell responses (referring to document Kishore A.H. etc., Biochem.J. (2007) BJ20070390; Banerjee, S. etc., Mol.Cell Biol. (2004) 24:2052-2062).The activity of PC4 is subjected to the further adjusting of post translational modification, and the type of this post translational modification comprises phosphorylation modification and acetylation modification at least.PC4 is by after the phosphorylation modification, the activity inhibited of itself and the effect of target activity factor and can oppositely mediate the function of its co-activator.Mass spectrometry results shows: the super phosphorylation modification of intravital PC4 is mainly by casein kinase i I mediation and occur over just N-terminal filament propylhomoserin enrichment region (referring to document Ge etc., Proc.Natl.Acad.Sci.USA (1994) 91:12691-12695).The acetylation modification of PC4 is mediated and is subjected to the inhibition (Kumor, P.B.R. etc., J.Biol.Chem. (2001) 276:16804-16809) of phosphorylation modification by P300.

Up to the present, whether everybody not clear PC4 role in regulator gene is relevant with the generation of cancer or tumor.

Summary of the invention

The inventor has adopted the different model organism system that comprises xenopus leavis oocytes system, tissue, other mammal cell lines, be surprised to find PC4 have cancer protein function and cell differentiation, tumor grow and the process of pathological changes in play an important role, see the multiple function of the PC4 shown in Figure 10, all these functions, especially Figure 10 acceptance of the bid pours down the function of line, the basis of all relevant with pathogenesis of cancer mechanism or pathogenesis of cancer.Particularly, the inventor discovers in a lot of malignant tumor tissues, as in cancerous lung tissue, Bladder Cancer, colon cancer tissue, breast cancer tissue, endometrium cancerous tissue, thyroid carcinoma tissue, carcinoma of small intestine tissue, find that all PC4 is activated at protein level.On the contrary, the protein level of the PC4 in the normal structure that does not live through accordingly quick growth does not raise.In cancerous cell line and other transformation cell lines, also found the rising of PC4 rna level, but in primary cell line, do not found.

For example, the inventor has measured the expression of the PC4 in the development by metamorphosis process of Africa xenopus.Assay method is as follows: the whole lysate of collecting the Africa xenopus of different developmental phases, monitor the protein content of each lysate, choose the total protein in each stage lysate of 50ug, last SDS-polyacrylamide gel electrophoresis is analyzed, then the albumen on the gel is forwarded on the nitrocellulose membrane, carry out western blot analysis at last, promptly adopt the polyclonal antibody of anti-PC4 to detect the albumen that is transferred on the nitrocellulose membrane.Measurement result is shown in Fig. 5 A, Fig. 5 B and Fig. 5 C, and the expression of PC4 is relevant with Africa xenopus bufonid toad development by metamorphosis process, and when current development by metamorphosis began, PC4 was significantly activated in 56～58 these stages.

The inventor is relevant with cell death by transfection test experience proof PC4.After the coding region fusion with people PC4 coding region (people PC4 coding region wild type or serine enrichment region saltant) and green fluorescent protein (GFP), sub-clone is to including in the CMV promoter mammalian expression vector (pcDNA3.1) again, to obtain the synthetic vectors transient transfection again to HeLa cell (referring to Ge etc., Proc.Natl.Acad.Sci.USA (1994) 91:12691-12695).After the transfection 24 hours, observation sample under laser confocal microscope can be observed PC4 role in the HeLa of transfection cell.The result is shown in Fig. 6 A, Fig. 6 B, Fig. 6 C, Fig. 6 A is the fluoroscopic examination result of matched group (the HeLa cell of untransfected), the fluoroscopic examination result of the HeLa cell of Fig. 6 B is transfection wild type PC4, Fig. 6 C has been transfection N-terminal filament propylhomoserin region mutation type PC4 (PC4-Δ S, it can not be by phosphorylation, so should be activatory fully) the fluoroscopic examination result of HeLa cell.Three width of cloth figure contrast as can be seen, and PC4 is arranged in nucleus and can causes chromosome condensation and apoptosis, especially can judge that from Fig. 6 C the overexpression of PC4 has caused apoptosis, and this point is consistent with the effect of cancer protein.

In addition, the inventor also adopts the western blot analysis method to measure PC4 level in many tissues.The different tissue (being designated as of malignant tumor tissue " T ", normal structure is designated as " N ", is provided by NIH) of operation back excision is provided, and fast refrigeration in-80 ℃ of environment until use.Organize the preparation process of lysate as follows: after in organizing solution, adding lysis buffer (lysis buffer volume be cumulative volume 1/10), it to be uniformly dispersed with the miniature high-speed agitator.The solution part is collected in centrifugal back.Detect the protein concentration of collected solution part lysate, can adopt Bradfort method of protein detection (BioRad Protein Detection instrument).For measuring the expression of PC4, the lysate of organizing of getting 50ug normal structure or malignant tissue carries out the analysis of SDS-polyacrylamide gel electrophoresis, albumen on the gel is forwarded on the nitrocellulose filter, carry out western blot analysis at last, promptly adopt the polyclonal antibody of anti-people PC4 to detect the albumen that is transferred on the nitrocellulose membrane.The result is shown in Fig. 8 B, this result shows in a lot of body tumor tissues, as cancerous lung tissue, Bladder Cancer, colon cancer tissue, breast cancer tissue, endometrium cancerous tissue, thyroid carcinoma tissue and carcinoma of small intestine tissue, all find PC4, but in corresponding normal human tissue, all do not detected PC4.

The inventor also adopts immunohistochemical analysis to detect the proteic expression of PC4 in normal lung tissue and the cancerous lung tissue.The slide specimen of normal lung tissue that provides with NIH or cancerous lung tissue section is done immunohistochemical analysis.The immunohistochemical analysis process is as follows: handle the microscope slide of tissue slice earlier with dimethylbenzene, and 2 times, each 15 minutes; Then it is inserted the ethanol of (from 100%～70%) that concentration successively decreases successively, hatched 5 minutes at every turn; Oven dry is after 5 minutes down at 95 ℃, and water and PBS rinsing successively carried out mounting with 3% milk confining liquid then; Add one anti-(i.e. the antibody of anti-PC4), incubated at room 1 hour; Flush away unnecessary one anti-after, tangential section the two anti-of fluorescence of labelling, incubated at room 30 minutes; At last, the rinsing microscope slide is placed under the fluorescence microscope and can detects PC4 albumen.Shown in Fig. 9 A and Fig. 9 B, do not detect PC4 albumen in the normal lung tissue, and in all cancerous cell of cancerous lung tissue, all find the proteic coagulation of PC4.

The inventor (has detected the mRNA of PC4 at all transformation cell lines (cos, HeLa, 293) and three breast cancer cell lines after testing in (MCF7, MD231, MDA468), but do not detect the mRNA of PC4 in former generation NIH3T3 cell line and nonmalignant rat tissue (cerebral tissue m-Brain, testis tissue m-Testis).Detection method (Northern engram analysis) is as follows: extract total RNA and separate by formaldehyde-agarose gel of 1% from above-mentioned these different cell lines or tissue, after the RNA on the gel forwards on the nitrocellulose membrane, will be marked with again ³²The people PC4 DNA (h-PC4 probe) of P and people p52 DNA (h-p52 probe) are hybridized (placing the probe solution environment to hatch a period of time film) with the total RNA that is extracted respectively as probe, detect the expression of PC4 mRNA and p52 mRNA among the total RNA of each sample respectively with ethidium bromide (EtBr) staining, i.e. 28S after the observation dyeing and 18S ribosomal RNA (referring to document Ge etc., EMBO J. (1998) 17:6723-6729).Testing result is shown in Fig. 7 A, Fig. 7 B, Fig. 7 C.Described p52 is the another kind of transcriptional coactivator that exists in above-mentioned various cell lines and tissue.

The inventor also with PC4 in tumor tissues activation and the activation of DNA topoisomerase I further connect, shown in Fig. 8 A, Fig. 8 B.Described DNA topoisomerase I is a kind of enzyme that DNA is untwisted and is some cancer therapy drugs such as NB-506, J-I09, and J-382, the target spot of DB-67 (referring to document Pilch, B. etc., Cancer Res. (2001) 61:6876-6884; Chen, A.Y. etc., Mol.Cancer Thera. (2005) 4:317-324).

Many oncogenes and tumor suppressor gene encoded protein, for example C-myc, P53, DNA topoisomerase I, also be everybody know can be as the albumen of transcription regulaton factor, the inventor finds that after deliberation PC4 can improve these proteic activity.

The above fact can draw a conclusion: except playing the effect of transcriptional coactivator, the people transcribes positive cofactor 4 (PC4) can also come active cell growth, differentiation, vicious transformation and tumor invasion growth as a kind of cancer protein.In fact, some adopt in other researchs of DNA array approach, have found that also PC4 is at activation (Das, C. etc., Mol.Cell.Biol. (2006) 26:8303-8315 of rna level in the cancerous lesion process; Kleivi, K. etc., Mol.Cancer (2007) 6:1-16).

Based on found that PC4 can play an important role as a kind of cancer protein in the forming process of dissimilar people's tumors, the inventor is the method that the research target spot has been invented a series of cancer diagnosis with PC4, prevention, treatment method for cancer and medicine, and the method for screening cancer therapy drug.

The invention provides the method for diagnosis experimenter's cancer or other malignant tumor, this method comprises following content: collect suitable sample or the liquid-like organized on one's body from the experimenter, detect in the collected sample expression as the PC4 of biomarker, if the expression of PC4 wherein comparison height in the same old way illustrates that sample is cancerous tissue or malignant tissue.For further making a definite diagnosis, this method can also include the step of the level that detects other relevant biomarkers, for example detects the expression of DNA topoisomerase I.

The present invention further provides a kind of experimenter of detection and whether suffered from the method that whether has malignant cell in cancer or the body, this method comprises following content: collect suitable sample on one's body from the experimenter, detect in the sample expression as the DNA topoisomerase I of biomarker, if the level of DNA topoisomerase I wherein comparison height in the same old way illustrates that sample is cancerous tissue or malignant tissue.

Above-mentioned " experimenter " comprises the people, other may cancered animal.

The invention provides a kind of function and prevent or treat the medicine of cancer, comprised the suitable PC4 antagonist of effective dose in this medicine by PC4 level in the reduction cell or inhibition PC4.

Accordingly, according to inventive concept of the present invention, the present invention also provides a kind of treatment method for cancer, uses the above-mentioned medicine that has comprised the suitable PC4 antagonist of effective dose promptly for the experimenter who suffers from cancer.Studies show that through the inventor this class medicine corresponding methods of treatment in other words can be used to treat pulmonary carcinoma, bladder cancer, colon cancer, breast carcinoma, carcinoma of endometrium, thyroid carcinoma, carcinoma of small intestine and other malignant tumor (referring to Fig. 8 B).

The above-mentioned medicine that includes suitable PC4 antagonist not only can also can be used to prevent purpose owing to treatment, prevents the generation of people's tumor.

Above-mentioned treatment method for cancer comprises chemotherapy, radiotherapy, immunotherapy and Comprehensive Treatment.

The medicine of above-mentioned prevention or treatment cancer can use, also can unite other one or more cancer therapy drugs separately and use, and reaches the effective dose of prevention or treatment cancer jointly.

Can add the carrier of pharmaceutically accepting in the medicine of above-mentioned prevention or treatment cancer.

Based on the prior biological theory and technology, some suitable PC4 antagonisies have been the present invention further provides: the antibody of PC4 polypeptide that modified or non-functional or albumen, anti-PC4, suitable antisense nucleoside acid molecule, suitable small molecules interference RNA molecule (siRNA).

PC4 polypeptide that modified or non-functional or albumen

That above-mentioned PC4 antagonist can be selected to have modified or do not have function PC4 polypeptide or albumen.PC4 is a kind of multifunctional protein of called optical imaging, verified its binding DNA and with other albumen interactional ability takes place in binding DNA.Everybody has recognized that a kind of PC4 mutant that modified or non-functional or part fragment wherein may be suppressed by PC4 wild type or unmodified institute dominance, perhaps may be present in the cell with recessive state at present.Perhaps, mutant that modified or non-functional, analog or part fragment wherein may with the protein competition of wild type or unmodified, thereby weaken the proteic function of wild type or unmodified effectively.For example, because the PC4 of phosphorylation is inactivation (the especially PC4 inactivation of N-terminal filament propylhomoserin enrichment region after by phosphorylation modification), it also can be used to unphosphorylated PC4 (activity form) thereby competition reaches therapeutic purposes; In addition, studies show that at present, wherein include the phenylalanine single amino acids sudden change (F77P) in the 77th site or lost the activity of binding with DNA with the PC4 albumen or the polypeptide of the sudden change of this sudden change equivalence, therefore according to inventive concept of the present invention, can infer that this no function PC4 polypeptide or albumen have the pharmaceutical use of anticancer aspect.They have the simulating peptide or the simulating peptide compositions of this similar functions may become the treatment of cancer medicament that a kind of urgent need remains to be researched and developed, owing to can prevent that immune system of animal or Proteometabolism mechanism from being destroyed.

The present invention also further provides some saltant PC4 albumen as PC4 antagonist (treatment depends on the medicine of the tumor of PC4).

Reported N-terminal filament propylhomoserin enrichment region is essential for the proteic activity of PC4 before, and is subjected to the adjusting of the phosphorylation of tyrosine kinase II, and deletion N-petiolarea causes the proteic auxilliary activation of PC4 to completely lose (Ge etc., 1994a; 1994b; Kretzschmar etc., 1994).The inventor has located some important amino acids that are absolutely necessary by making a series of single amino acids sudden change concerning the proteic activity of PC4.Thus, the inventor filters out 7 kinds of saltant PC4 albumen, as shown in Figure 2, mt-1 (K23I/K29A), mt-2 (K35I/K41A), mt-3 (R27A/K28I/K29A), mt-4 (R47N/K35I/K41A), mt-5 (F77P), mt-6 (K29A), mt-7 (K41A).Prove through experimentation, the PC4 that single amino acids sudden change (F77P) has taken place except known the 77th site has lost ability and the transcriptional activity in conjunction with DNA, the proteic activity of PC4 that dual sudden change (K23I/K29A and K35I/K41A) and triple mutant (R27A/K28I/K29A) take place at N-end group local area also greatly reduces, therefore PC4 mutant: F77P, K23I/K29A, K35I/K41A, R27A/K28I/K29A can be used as cancer therapy drug, reach the effect that treatment depends on the malignant tumor of PC4 by competing with endogenous wild type PC4.

The present invention also provides short polypeptide with the amino acid sequence homologous in the activation structure territory of PC4 as the PC4 antagonist.

PC4, as a general transcriptional coactivator, its activity shows as and other general transcription factors, activating transcription factor, dna profiling between interaction, so the activation structure territory realizes that for PC4 its function is necessary.Thus, the inventor designs and has synthesized two polypeptide, and their sequence is consistent with two PC4 activation structure domain amino acid sequences respectively, and these two polypeptide can combine other with wild type PC4 competition and activate son, thereby reaches the active effect of PC4 that suppresses.These two peptide sequences are: 16-30 aminoacid (DSDSEVDKKLKRKKQ sees SEQ ID NO.3) and 26-40 aminoacid (KRKKQVAPEKPVKKQ sees SEQ ID NO.1) sequence in the aminoacid sequence of PC4.

The present invention also provide can with the bonded simulation activation structure of PC4 domain polypeptide as the PC4 antagonist.

Because the carcinogenic activity of PC4 is (specificity of PC4 and described oncogene, somatomedin or other cancerigenic factors activates the son interaction and activates their expression) of realizing by its expression as the whole or most oncogene of transcriptional coactivator deexcitation, somatomedin or other cancerigenic factors, therefore, suppressing PC4 albumen goes can make PC4 albumen lose its auxilliary son activity that activates in conjunction with endogenous activation.The inventor has found that after deliberation has 15 amino acid whose amphipathic helix polypeptide (amphipathic helix peptide, AH peptide) can simulate a kind of activation structure territory and PC4 strong interaction takes place, thereby sealed the activation minor structure territory of PC4, suppress the effect of PC4 and endogenous activation, suppress the function of PC4, reached therapeutic purposes.The sequence of this polypeptide is: ELQELQELQALLQQQ, see SEQ ID NO.5.

As a further improvement on the present invention, the PC4 antagonist also comprises polypeptide or the protide PC4 antagonist that is connected with the migration domain.

In order to promote polypeptide or protide PC4 antagonist to enter tumor cell, make it to examine interior cancer protein PC4 by targeting, can by will move domain (translocation domain, TLD) with recombinant monoclonal antibodies, saltant PC4 albumen, with the polypeptide of the amino acid sequence homologous in the activation structure territory of PC4, can suppress fusions such as active albumen of PC4 or polypeptide with PC4 bonded simulation activation structure territory or other and realize.

The present invention adopted a kind of include 12 amino acid whose short polypeptide as the migration domain, its sequence is: PLSSIFSRIGDP, see SEQ ID NO.7, molecular weight is 1288.47, isoelectric point, IP pI=6.27.It is reported this peptide species (Oess and Hildt 2000 that for the infection of hepatitis B virus and some other proteic cell permeabilities, is absolutely necessary; Stoeckl et al., 2006).

Antibody

The invention provides the neutralizing antibody that some can suppress PC4 albumen or suppress the biologic activity of PC4 albumen and DNA topoisomerase I (these two also claim target protein below the albumen).The anti-PC4 antibody of antibody of the present invention for accessing based on existing biology techniques, preferred monoclonal anti PC4 antibody also can select to include the polyclonal antibody of described monoclonal anti PC4 antibody.Described monoclonal anti PC4 antibody has comprised the subtype specificity antibody of PC4.

According to inventive concept of the present invention, above-mentioned monoclonal anti PC4 antibody be can specificity in conjunction with the antibody of target protein, Fab in the antibody also claims the antigenicity fragment, and the antigenicity fragment can be Fab fragment, F (ab) 2 fragments, Fab ' fragment, F (ab ') 2 fragments, Fd fragment, Fd ' fragment or Fv fragment.Comprised the antigenicity fragment in the general monoclonal anti PC4 antibody, monoclonal anti PC4 antibody also can be selected these antigenicity fragments (being univalent antibody) certainly.Preferably select epitope, promptly in antibody enters body after by the site of antigen recognition.The first-selected mouse monoclonal antibody of monoclonal anti PC4 antibody or wherein antigenicity fragment, epitope.Except univalent antibody, domain antibodies (dAbs) (list of references Holt etc., Trends in Biotechnology (2003) 21:484-490) also is applicable to anti-PC4 antibody of the present invention.

If the treatment people should adopt humanized antibody, preferably the humanization natural antibody.

The method of making the antibody of known antigens has a variety of, this is that technology that those skilled in the art know is (referring to (the Cold Spring Harbor Laboratory Press of Cold Spring Harbor Laboratory publishing house, Cold Spring Harbor, NY) " the Antibodies:A Laboratory Manual " of Chu Baning, Harlow and Lane, 1988; Or referring to No. the 01/25437th, international monopoly).

Can adopt immunization in the body, come immune people or other animal reservoirs, from the animal reservoir, extract antibody again with the immunogenic component that has that is derived from the target protein; Can obtain above-mentioned antibody by immune people or the animal except that the people, preferred mammal, for example rat, mice, Cavia porcellus, rabbit, goat, sheep, pig, perhaps birds (as chicken), further preferred mice.

Also can adopt the mode of external immunity from immunocyte, to extract antibody.External immunization preferably adopts recombination and expression techniques to produce antibody, and the cell line that for example DNA with the anti-PC4 antibody of suitable coding transforms, transfection, infection or transduction are fit to obtains recombination system and can express the anti-PC4 antibody of production; Also can adopt hybridoma technology known in the art to prepare antibody;

Or adopt biological mode of rebuilding, directly with the heavy chain and the light chain of purification are built into antibody;

Particularly, can also select the method for chemosynthesis to obtain suitable antibody, perhaps adopt the method (for example the intracellular antibody technology is expressed anti-PC4 antibody in born of the same parents) of intracellular immunity.

In addition, antibody of the present invention has also comprised chimeric antibody and hybrid antibody.Technology (Morrison etc., Proc.Natl.Acad.Sci.USA, (1984) 81:6851-6855 of preparation chimeric antibody and hybrid antibody have been described in some documents; Neuberger etc., Nature, (1984) 312:604-608; Takeda etc., Nature (1985), 314:452-454).

Further, single-chain antibody also be applicable to the present invention (be two United States Patent (USP)s of Huston referring to the invention people: the 5th, 476, No. 786 and the 5th, 132, No. 405; Huston etc., Proc.Natl.Acad.Sci.USA, (1988) 85:5879-5883; The invention people is the 4th, 946, No. 778 United States Patent (USP)s of people such as Ladner; Bird, Science. (1988) 242:423-426; Ward etc., Nature. (1989) 334:544-546).Described single-chain antibody is a kind of small molecular antibody of being made up of single peptide chain, synthetic method: between heavy chain of antibody and variable region of light chain (Fv) gene, with one section small peptide gene both are connected into the ScFv gene, this expression of gene product can be folded into the single chain polypeptide with antigen binding capacity.

The route of administration of antibody can adopt drug conveying approach more well-known to those skilled in the art, and for example: direct injection may relatively be fit to antibody is discharged into the site that needs treatment.Also can adopt the liposome that is enclosed with antibody in its film, the liposome orientation is transported to target spot, thereby the expression of the target gene here or function are suppressed.Except monoclonal anti PC4 antibody, can also comprise some other medicaments in the described liposome, for example above-mentioned some can be released to the medicament (referring to document, Wolff etc., Biochem.et Biophys.Acta, (1984) 802:259) of treatment target spot.

The antisense nucleoside acid molecule

Also provide employing antisense oligonucleotide molecule to suppress the method for the expression of PC4 gene or inhibition PC4 gene and DNA topoisomerase I gene (these two genes are hereinafter to be referred as target gene) in the present invention.Well known in the art, the expression of the antisense RNA in the cell, particularly constructive expression, expression that can suppressor gene (may be or prevent the expression that the mode of montage is come suppressor gene) by the blocking-up translation.Based on this point, disturb montage to make that those conservatives are relatively poor and produce stronger specific intron sequences and can play a role, thereby can suppress the expression of corresponding gene product in the species, but not suppress the expression of the congener of this gene outcome in other species.

Term " antisense component " is meant and an abundant complementary RNA of specific mRNA molecule or dna molecular.Antisense rna molecule is special, and antisense RNA and mRNA molecular hybridization take place can cause the translation of mRNA to be suppressed.Described molecular hybridization environment in vivo takes place down.Antisense rna molecule of the present invention must have enough and the complementary ability of target gene, about 15～30 nucleotide of length, so antisense rna molecule can suppress the expression of target gene with target gene (perhaps mRNA) hybridization, no matter hybridizes to occur in montage level, transcriptional level or translation skill.Antisense rna molecule of the present invention select can with the RNA molecule of any one part hybridization that comprises coding region sequence, 3 ' or 5 ' non-coding area sequence or other intron sequences among the target cDNA, or can with the RNA molecule of target mRNA hybridization.As for how designing antisense nucleic acid molecule, on the basis of known mRNA sequence, according to prior art, the antisense nucleic acid molecule sequence can design at an easy rate.

Antisense rna molecule of the present invention, can adopt the mode of conversion or transfection, transport by carrier and to enter host cell, described bearer type comprises retroviral vector and plasmid, connect the DNA of encoding antisense RNA and the suitable adjusting sequence that includes promoter, together insert in the described carrier after the connection, thereby antisense RNA can be expressed in host cell.In one embodiment, the segmental carrier of cDNA that includes encoding antisense RNA molecule has been realized stable transfection and constructive expression, and perhaps this expression may be to realize under the control of the promoter of tissue specificity or development-specific.Also can use liposome that antisense rna molecule is transported and enter cell.

At interior therapeutic, present existing prefered method is directly antisense rna molecule to be transported to target spot, rather than has made up the segmental expression vector of cDNA of encoding antisense RNA molecule on it by stable transfection.Antisense oligonucleotide molecule of the present invention has the length of 15 to 30 bases, its sequence has determined whether this antisense oligonucleotide molecule can hybridize with any one part that comprises coded sequence, 3 ' or 5 ' noncoding region or other intron sequences among the target cDNA, perhaps preferred, whether can hybridize with target mRNA.Preferably select for use those to have the sequence of the strongest antisense ability with the sequence of the antisense oligonucleotide molecule of target gene hybridization.Antisense ability on the antisense oligonucleotide sequence molecule is decided by following factor: the accessibility of the length of antisense oligonucleotide, binding affinity, target sequence.At the strong antisense oligonucleotide molecule of in-vitro screening antisense ability, can be by vitro detection its suppress the ability of the ability of target protein translation phenotype relevant with target gene with inhibition, for example, the ability of cell proliferation in the inhibition culture medium.In general, most of zones of target cDNA (5 ' and 3 ' noncoding region, AUG sintering, coding region, splice junction and intron sequences) can both utilize antisense oligonucleotide to locate.

Antisense oligonucleotide molecule of the present invention is preferred, and those are stable, that strong nuclease-resistant ability is arranged, have the oligonucleotide molecule that suitable pharmacokinetics performance makes it arrive destination organization and to have the ability to pass plasma membrane with non-toxic.

According to prior art, the chemical modification of antisense rna molecule mainly concentrates on phosphodiester backbone, base, sugar ring, and these modifications can improve the stability of antisense rna molecule or the ability of hybridizing with target cDNA or mRNA.The preferred phosphorothioate antisense oligonucleotide of antisense oligonucleotide molecule of the present invention molecule (phosphodiester bond of antisense oligonucleotide molecule by the phosphorothioate modification then obtain phosphorothioate antisense oligonucleotide molecule).The basic structure of phosphorothioate antisense oligonucleotide molecule also can further be optimized its antisense ability by modification.In addition, had been found that also N3 '-P5 ' phosphoramidate bond energy enough makes the oligonucleotide molecule stablize and can improve the binding adhesion between antisense oligonucleotide molecule and the RNA in the presence of nuclease.Peptide nucleic acid(PNA) key (PNA) has all replaced ribose and phosphodiester backbone can be stablized it in the presence of nuclease, can be by RNA H enzyme action, and improved affinity between antisense oligonucleotide molecule and target gene or the mRNA.About the modification of base, verified, base can strengthen the antisense ability of antisense oligonucleotide molecule and make the antisense oligonucleotide molecule can not degraded by RNA H after being modified by some heterocycles, and for example, the C-5 thiazole is modified.At last, also can consider the modification of sugar ring, for example use 2 '-oxygen-propane base, 2 ' methoxyethoxy to replace ribose and make oligonucleotide can keep stability in the cell in vitro culture medium or in the intravital environment nuclease.

The route of administration of antisense nucleic acid oligomer molecule of the present invention should select some that the approach of best antisense effect (measuring according to above-mentioned standard) can be provided.Show with cationic-liposome, retroviral vector to be that carrier carries the drug effect of antisense nucleic acid oligomer or direct administration better with the testing in vitro result in the body of antisense nucleic acid oligomer molecule.Another kind of possible mode of administration be utilize can with the bonded antibody of target cell surface specific labelling with the molecular targeted purpose cell that is transported to of antisense nucleic acid oligomer.Antibody combine with target cell or with its on receptor combine the purpose that can both reach targeted.

Small molecules interference RNA

Also further provide the small molecules interference RNA that can suppress target gene and express (small interfering RNAs is called for short siRNA, also claims RNAi, also claims the RNA interfere RNA) in the present invention.Small molecules interference RNA is a kind of double stranded rna molecule, and typical length is 21 nucleotide (nt), if itself and target gene homology, then can the jamming target expression of gene.

The RNAi technology is relevant with the sequence-specific posttranscriptional gene expression process in the eukaryotic cell.In general, relate to the mRNA degraded of particular sequence in this process, this is degraded by being caused with the homologous double-stranded RNA of this section particular sequence (dsRNA).For example, can make the transmission of hereditary information become unstable with the expression of specific strand mRNA (ss mRNA) the corresponding long dsRNA of sequence, thereby disturb the expression of corresponding gene.Introducing all can suppress this expression of gene with the corresponding dsRNA of whole or most sequences of any selected genes mRNA.It seems that according to present research when a segment length dsRNA expressed, it is at first become length by the rnase iii enzyme action only was the short dsRNA of 21～22 base pairs.Therefore, siRNA can introducing or the expression of homologous short dsRNA be subjected to direct influence with it because of these.

Have at least two approach to be subjected to the influence of dsRNA in the mammalian cell.

In the sequence-specific approach, aforesaid, initial dsRNA is a plurality of small molecules interference RNAs of digested one-tenth (siRNA) earlier.These siRNA are made up of positive-sense strand and antisense strand that length is about 21 nucleotide, and every chain is made up of 19 nucleotide and 3 ' two free nucleotides holding that play interference effect.The sequence information that small molecules interference RNA is considered to provide certain makes the mRNA of respective specific sequence be degraded by targeting.

On the contrary, and non-specific approach can be caused by the dsRNA of arbitrary sequence, as long as this dsRNA length is at least greater than 30 base pairs.The initiation of non-special approach is because dsRNA has activated two enzymes: PKR enzyme and 2 ', 5 ' oligoadenylate synthetase (2 ', 5 '-AS).When the PKR enzyme is in activated state, can close all proteic synthesizing by phosphorylation translation initiation factor eIF2; 2 ', 5 ' oligoadenylate synthetase can synthesize the molecule of a kind of activator RNA enzyme L (RNase L), and RNase L is the non-specific enzyme of a kind of all mRNA that can degrade.Non-specific approach is represented host's reaction of pressure and viral infection to external world usually, and in general, the effect of non-specific approach is preferably dropped to floor level.Clearly, long dsRNA can only induce non-specific approach, therefore, the dsRNA of 30 base pairs of curtailment preferably by RNA disturb (RNAi) be the sequence-specific approach influence expression of gene (referring to Hunter etc., J.Biol.Chem. (1975) 250:409-417; Manche etc., Mol.Cell.Biol. (1992) 12:5239-5248; Minks etc., J.Biol.Chem. (1979) 254:10180-10183; Elbashir etc., Nature (2001) 411:494-498).

Verified, it is a kind of effective means that reduces gene expression dose that RNA disturbs, and the cell of most of types all is suitable for, for example HeLa cell, NIH/3T3 cell, COS cell, 293 cells and BHK-21 cell.Particularly compare with adopting antisensenucleic acids, siRNA can drop to destination gene expression more low-level, and in fact, siRNA often can make genes of interest not express (referring to Bass, Nature (2001) 411:428-429) fully.In addition, for reducing destination gene expression level in the mammalian cell, under the equal conditions, the effective dose of siRNA is than the low several magnitude (Elbashir etc., Nature (2001) 411:494-498) of effective dose of antisense RNA.

RNA disturbs the double-stranded oligonucleotide of preferred length less than 30 base pairs, and further preferred length is the double-stranded RNA of 25,24,23,22,21,20,19,18,17 base pairs.Length is that the siRNA effect of 21 base pairs is better.It is better to include free 3 ' end siRNA effect.Typical free 3 ' end has two free nucleotide, these two nucleotide can be made up of the ribonucleotide residue of any kind, preferred 2 '-deoxyribonucleotide, in cell culture medium and in the transfectional cell, adopt 2 '-free 3 ' end that deoxyribonucleotide is formed not only reduced the synthetic cost of RNA, and can strengthen the ability (referring to Elbashi etc., Nature (2001) 411:494-498) of siRNA opposing nuclease enzyme action.

Longer dsRNA molecule for example has the macromole dsRNA of 50,75,100 or even 500 or more base pair, also can be applied in specific embodiment of the present invention.For the normal concentration that reaches the dsRNA that the RNA interference effect adopted is about 0.05nm, 0.1nm, 0.5nm, 1.0nm, 1.5nm, 25nm or 100nm, though also can adopt other concentration, this depend on handled cell type, at target gene and other correlative factors.

Synthesizing of longer dsRNA molecule: can be from the synthetic long-chain dsRNA of promoter transcription, described promoter is the t7 rna polymerase promoter as known in the art.Long dsRNA molecule synthetic that suppresses the single target gene: light from the target bit in the promoter downstream of cell-free transcription folder, synthesize complementary RNA molecule simultaneously to two different directions, and be assembled into corresponding dsRNA immediately.State these dsRNA in design and divide the period of the day from 11 p.m. to 1 a.m, must consider that each RNA molecule all should include a part and the homologous nucleotide sequence of target gene mRNA.

The basic synthetic method of siRNA: adopt chemical method to synthesize or make up suitable expression vector and in external or body, synthesize.The synthetic standard rna of chemical method is made up of 21 ribonucleotides.Synthetic siRNA can adopt the gel separation method to carry out purification.(Elbashir etc., Genes Dev (2001) 15:188-200).

When design siRNA sequence, can select any one section nucleotide sequence adjacent as specific sequence with the coded sequence of target gene mRNA, that is to say, can select when designing siRNA of the present invention with PC4mRNA on the adjacent any one section nucleotide sequence of coded sequence as specific sequence.In addition, how from these nucleotide sequences, to have selected best specific sequence? general some existing sequential design softwares that adopt.At first utilize the secondary structure of these software program target of prediction gene mRNAs, select the sequence that those appear at the exposed strand district on the folding mRNA probably again.Design method that is adopted and the material of suitable siRNA, can be with reference to United States Patent (USP) the 6th, 251, the corresponding contents of putting down in writing in No. 588 files.

Though mRNA is considered to linear molecule usually, comprised in its nucleotide sequence and directly instructed the synthetic information of albumen, in fact, most mRNA demonstrates and includes a lot of secondarys and three grades of stereochemical structures.Secondary building unit on the RNA is to form by taking place between the zones of different in the same RNA molecule to interact, and described interaction mainly is the interaction of Watson-Crick type.Important secondary building unit comprises double-spiral structure, hairpin structure and the prominent ring structure that is formed by intramolecularly in double-stranded RNA (dsRNA) and the internal ring district.Formed more complicated three dimensional structure is the tertiary structure unit when generation interaction or secondary building unit and strand district have an effect between the secondary building unit.Many researchers measure in a large amount of RNA duplex structures in conjunction with energy, thereby drawn and a series ofly can be used for predicting that the rule of RNA secondary structure is (for example referring to Jaeger etc., Proc.Natl.Acad.Sci.USA (1989) 86:7706; Turner etc., Annu.Rev.Biophys.Biophys.Chem. (1988) 17:167).These rules can help identification mRNA construction unit, and particularly for the exposed strand zone of identification, the exposed strand zone on these mRNA may become the best target fragment of siRNA, ribozyme or antisense RNA institute targeting.Accordingly, the best target fragment on the identification PC4 mRNA can be used for designing based on the dsRNA oligonucleotide with RNA interference effect of the present invention's design, suitable ribozyme and tup forming core enzymatic compositions.

The above-mentioned dsRNA oligonucleotide with RNA interference effect can connect the same allogenic target gene mode transfered cell by the support agent transfection together, and described support agent is the lipofectamine Lipofectamine 2000 at attached cell system that produces of the Life Technologies company of liposome-for example as known in the art.The lipofectamine Oligofectamine that the transfection carrier of the dsRNA oligonucleotide of targeting endogenous gene can select for use Life Technologies company to produce.Transfection efficiency can also detect in the following way: is after cotransfection is expressed with dsRNA oligonucleotide and hGFP-encoding pAD3 at mammalian cell, utilize fluorescence microscopy to detect its transfection efficiency (referring to Kehlenback etc., J.Cell.Biol. (1998) 141:863-874).Behind the dsRNA transfered cell, its RNA jamming effectiveness can adopt a lot of methods to detect.These methods comprise western blot analysis (Western blot analysis), RT-polymerase chain reaction (RT-PCR) and Northern hybridization analysis (Northern blot analysis), described western blot analysis is meant that waiting for that the endogenous gene storehouse is finished once has enough to meet the need metabolism, new round albumen is synthetic be suppressed after, the method that adopts antibody to come recognition objective gene outcome (PC4); RT-polymerase chain reaction and Northern hybridization analysis can be measured the level of target gene mRNA (PC4 mRNA).

The reagent that is adopted about the RNAi technology, methods and applications have further introduction in No. the 6278039th, 5723750 and 5244805, United States Patent (USP), being listed in herein can be for referencial use.

Another object of the present invention provides the screening technique of cancer therapy drug, and this method is based on PC4 to be a kind of cancer protein and to can be used as this scientific discovery of cancer therapy drug target spot.

Screening technique based on promoter

The method of screening cancer therapy drug of the present invention, this method comprise following two steps that take place successively: (1) provides a kind of gene construct, and it comprises the promoter sequence of PC4 gene and the reporter gene that effectively is connected with this promoter sequence; (2) drug candidate and described gene construct are mixed the environment that places suitable described reporter gene expression, wherein, the drug candidate that suppresses reporter gene expression is a cancer therapy drug.

The PC4 gene promoter sequence is referring to Figure 1A.With the suitable reporter gene that this promoter sequence effectively is connected, can select as luciferase gene or green fluorescence protein gene.Those skilled in the art should be able to recognize, the exemplary sequence that in present specification, is provided, also have many suitable PC4 gene promoter sequences or other cis acting transcription factor of some PC4 genes also can be applied to the method for screening anticancer medicine of conceiving based on the present invention.

Except the above-mentioned screening technique based on promoter, method for screening anticancer medicine of the present invention can also adopt the detection method based on protein active.

Depend on the proteic transcriptional activity detection method of PC4

The present invention also provides a kind of method of screening cancer therapy drug, and this method comprises following two steps that take place successively: (1) provides a kind of PC4 of detection the test of proteic transcriptional level; (2) add drug candidate in above-mentioned test, wherein, the drug candidate that reduces the PC4 transcriptional level is a kind of cancer therapy drug (referring to Ge and Roeder, Cell (1994) 78:513-523).

Detection method based on protein-protein or protein-DNA interaction

The present invention also provides the method for another kind of screening cancer therapy drug, and this method comprises following two steps that take place successively: (1) provides and detects between PC4 and the albumen interactional test between interaction or PC4 and the DNA; (2) in above-mentioned test, add drug candidate, wherein, reduce between PC4 and the albumen between interaction or PC4 and the DNA interactional candidate compound and be cancer therapy drug (referring to Ge, Nucleic Acids Res. (2000) 28, e3).

Preferred high throughput system (HTS) screening suppresses the chemical compound of PC4 protein active.

In high flux screening detects, usually (fluorescence resonance energy transfer FRET) comes molecular dynamics (for example protein-protein interaction, protein-dna interaction, protein conformation conversion etc.) to carry out quantitatively to use the FRET (fluorescence resonance energy transfer) technology.General 96 orifice plates or the 384 orifice plate standards of adopting of high flux screening detection system based on the FRET (fluorescence resonance energy transfer) technology.

Screen the chemical compound of inhibition PC4 protein active of the present invention and use amphipathic helix (AH) the domain polypeptide that is marked with fluorescence as energy donor, the PC4 albumen of mark fluorescent is as energy acceptor.In case PC4 and amphipathic helix domain polypeptide form complex, promptly owing to the interaction force between two molecules makes that distance is very near (apart from about 1～10nm) between donor and the receptor, what observed this moment mainly is the emission light of PC4, because intermolecular fluorescence resonance energy is transferred to PC4 albumen from amphipathic helix domain polypeptide.The exciting light of amphipathic helix domain polypeptide and the emission light of PC4 are subjected to the monitoring that fluorescence intensity is read the plate instrument.The effect of the inhibition PC4 protein active of any chemical compound can both be measured by the variation of monitoring the fluorescence intensity that is read.General screening two compounds: 1) micromolecule synthetic; 2) native compound.Certainly, these chemical compounds also need further be confirmed, prove that it can become candidate's anticarcinogen that a kind of potential treatment depends on the malignant tumor of PC4.

The method of cancer diagnosis of the present invention or screening cancer therapy drug is based on protein or based on the detection method of nucleic acid, these methods all are conventional methods well-known to those skilled in the art.Anti-PC4 antibody and anti-topoisomerase I antibody all are antibody that some are known and that can buy on market.For example, with the antibody of nano Au particle covalent cross-linking, can buy from BioassayWorks company (Ijamsville, Maryland), the enough levels of monitoring proteic level of PC4 or topoisomerase I apace quantitatively of this antibody capable.Same, reverse transcriptional PCR (RT-PCR) can be monitored the activation levels of PC4 and topoisomerase I at rna level.

In sum, the inventor has found that PC4 is relevant with cancer, opened up the anticancer research frontier, and invented a series of Method for cancer diagnostics and reagent (based on the expression that detects PC4) thus, and the method for treatment and prophylaxis of cancer with medicine (based on the function and the expression that suppress PC4), screen the method for cancer therapy drug etc.

Description of drawings

Figure 1A, Figure 1B, Fig. 1 C provide the sequence information of people PC4 gene; Wherein, Figure 1A provides people PC4 cDNA sequence and promoter region, and promoter region has marked underscore (referring to the GenBank serial number: NM_006713); Figure 1B provides the DNA sequence of people PC4 coding region; Fig. 1 C provides the aminoacid sequence of people PC4 albumen (127 aminoacid), and the 77th aminoacid (F: phenylalanine has been marked underscore) site can make PC4 lose and the bonded ability of single stranded DNA if the single amino acids sudden change takes place;

Fig. 2 shows PC4 N-petiolarea mutation analysis collection of illustrative plates, and described N-petiolarea comprises base region and the 77th aminoacid; Wherein, WT represents wild type, and mt represents saltant, and mt-1～7 have been represented 7 different saltant PC4 albumen of the present invention respectively;

Fig. 3 A, Fig. 3 B, Fig. 3 C show the functional analysis collection of illustrative plates of PC4 mutant (mt-1～7); Wherein, Fig. 3 A shows the proteic standardized testing of reorganization PC4 of various purification; Fig. 3 B shows various proteic electrophoretic mobility shift assay (EMSA) experimental result that Fig. 3 A is detected, and it is active with combining of dsRNA promptly to detect each albumen), the PC4+DNA among the figure is meant the complex of PC4 and dsDNA; Fig. 3 C shows the various proteic in vitro transcription test experience result that Fig. 3 A is detected, and wherein pG5HM is the template of transcribing that depends on PC4, and pML Δ 53 is the templates of transcribing that do not rely on PC4;

Fig. 4 A, Fig. 4 B, Fig. 4 C, Fig. 4 D, Fig. 4 E, Fig. 4 F show wild type PC4 albumen and the proteic protein arrays analysis result of various saltant PC4; Wherein 5 and 40 of the collection of illustrative plates left side represented two different protein concentrations (pmol of unit), Fig. 4 C shows the interaction result between each albumen and the transcription factor TFIIA (p55); Fig. 4 E shows the interaction result between each albumen and the transcription factor Spl; Fig. 4 F shows the interaction result between each albumen and the ssDNA; Fig. 4 A shows Ponceaux (Ponceau S) coloration result, and whether this experiment is to detect each albumen successfully to forward on the nitrocellulose membrane; Whether the interaction result that Fig. 4 B shows between anti--PC4 antibody (anti-PC4) and each albumen, this experiment are consistent in order to detect each the proteic amount that forwards on the film; Fig. 4 D is the blank result, detects between each albumen and the Gal4 (1-94) whether non-specific binding is arranged;

The presentation of results of Fig. 5 A, Fig. 5 B, Fig. 5 C the expression of PC4 and the relation of Africa xenopus development by metamorphosis; Every different development by metamorphosis stage of row gel band representative; Wherein, Fig. 5 A and Fig. 5 B show the expression of the PC4 in 2～58 and 56～64 each stage respectively, and " O " represented oocyte (oocytes) among Fig. 5 A, and " E " represented embryo (embryos), and PC4-P represents the PC4 albumen (inactivation) of phosphorylation; Fig. 5 C shows the relation of PC4 activation and Africa xenopus differentiation and development, wherein shows the expression curve of the expression curve of thyroxin (T3 and T4) and Thyroid Hormone Receptors (TR α and TR β) and PC4 expression curve in contrast;

Fig. 6 A is matched group (the HeLa cell of untransfected) fluoroscopic examination result; The fluoroscopic examination result of the HeLa cell of Fig. 6 B is transfection wild type PC4, the fluoroscopic examination result of serine region mutation type PC4 (PC4-Δ S) that Fig. 6 C has been transfection, illustrated as PC4 transfectedly in the Hela cell time, PC4 is arranged in nucleus and can causes chromosome condensation and cell death.

Fig. 7 shows the testing result of PC4 mRNA; Selected test sample is as follows: transformation cell lines (cos, HeLa and 293), three breast cancer cell lines ((MCF7, MD231 and MDA468), primary cell line (NIH3T3), rat normal structures (brain m-brain and testis tissue m-Testis); Fig. 7 A represents the abundance measurement result of total RNA in each sample; The expression (being the express spectra of p52) that Fig. 7 B shows p52 mRNA in contrast; Fig. 7 C shows the expression of PC4 mRNA;

Fig. 8 A and Fig. 8 B show the western blot analysis result of DNA topoisomerase I and PC4 in various tumor tissues and the corresponding normal structure respectively, wherein, Kidn (nephridial tissue), Lung (lung tissue), Bladd (bladder body), Colon (colon), Prost (prostata tissue), Breast (mammary gland tissue), Pancrs (pancreatic tissue), Endomt (endometrial tissue), Thyro (parathyroid tissue), S-Bow (small intestine), N represents normal structure, T represents malignant tissue's (tumor), PC4a represents to have active wild type, and PC4b represents to have active inferior wild type;

Fig. 9 A and Fig. 9 B show the immunohistochemical analysis result of PC4 expression in human body normal lung tissue and the cancerous lung tissue respectively;

Figure 10 shows the multiple function of PC4; These all functions especially mark the relevant with cancer of underscore or relevant with the pathogeny of cancer, so PC4 may become the treatment target spot of a lot of cancer therapy drugs.

The specific embodiment

Further set forth the present invention below in conjunction with embodiment and accompanying drawing.

Embodiment 1

Saltant PC4 albumen

The inventor has located some important amino acids that are absolutely necessary by making a series of single amino acids sudden change concerning the proteic activity of PC4.Thus, the inventor filters out 7 kinds of saltant PC4 albumen that can be used as the PC4 antagonist, collection of illustrative plates as shown in Figure 2, mt-1 (K23I/K29A), mt-2 (K35I/K41A), mt-3 (R27A/K28I/K29A), mt-4 (R47N/K35I/K41A), mt-5 (F77P), mt-6 (K29A), mt-7 (K41A).

The inventor has also carried out recombination to construct, expression, purification, evaluation with above-mentioned 7 kinds of saltant PC4 albumen, and is specific as follows.

The recombination to construct method of mutant: produce various sudden change construct (the double-stranded oligonucleotide that includes the specified point sudden change can commercial synthetic obtaining) thereby the PC4 wild type district in the bacterial expression vector is replaced as the double-stranded oligonucleotide that includes specified point sudden change.

Each sudden change construct all will carry out sequencing analysis and just can be identified after the expression in E coli.Expression PC4 albumen (comprising wild type and saltant) in E coli can obtain by 2 following step method of purification: the first step: P11 cellulose phosphate ion-exchange chromatography; Second step: heparin affinity chromatography.Proteic concentration can be measured by the Bradford method behind the purification, and purity can be passed through polyacrylamide gel electrophoresis (SDS-PAGE) and measure.PC4 albumen behind the purification (WT, mt-1～7) is gone up SDS-PAGE, and electrophoresis result as shown in Figure 3A.

Adopt following method to detect the proteic activity of PC4 behind the purification: 1) electrophoretic mobility shift assay experiment (the results are shown in Figure 3B); 2) in vitro transcription test experience (the results are shown in Figure 3C); 3) protein arrays analysis (the results are shown in Figure 4), these three kinds of method concrete operations are as follows:

1) (Electrophoresis mobility shift assay EMSA) detects the ability that PC4 albumen is bound dsDNA to adopt the electrophoretic mobility shift assay method

The operation of EMSA is as follows: configuration 20ul reaction system (wherein including the 10ng various PC4 protein samples of purification) and the length that is marked with 32P are the dsDNA probe (deriving from the adenovirus major late promoter district) of 64bp, the reactant mixture that both is mixed the back gained places 4 ℃ of incubations after 1 hour, last 8% polyacrylamide gel electrophoresis can detect mobile PC4-dsDNA complex by autoradiography.Shown in Fig. 3 B, mt-1, mt-2, mt-3, mt-5 have lost the ability with the dsDNA binding.

2) in vitro transcription test experience

Utilization from the HeLa cell or from a reorganization system general transcription factor of purification, as TFIIA, TFIIB, TFIID, TFIIF, TFIIH, rna plymerase ii wait to transcribe test experience at reconstruction in vitro.The activation of this in vitro transcription system depends on PC4 and other and activates son (referring to Ge etc., 1996).Except these above-mentioned general transcription factors, this system of transcribing also comprise template (pG5HM is the template of transcribing that depends on PC4) that a response activates son, one do not rely on the basic templates (pML Δ 53 is the templates of transcribing that do not rely on PC4) that activates son and ³²P-CTP.This is transcribed, and 30 ℃ of incubations extracted new synthetic RNAs after 1 hour in the system, it is carried out SDS-PAGE analyze autoradiography again.The result is shown in Fig. 3 C, and mt-1, mt-2, mt-3, mt-5 have lost PC4 albumen substantially as transcribing the auxilliary function that activates son.

3) protein arrays analysis experiment

Protein arrays analysis experiment is based on " general protein arrays " method of (also claiming UPA) (referring to document Ge 2000).A large amount of albumen of purification are being fixed on the nitrocellulose membrane by certain arrangement regulation, are forming intensive lattice array, the about 1mm of every spot diameter includes the albumen (depending on proteic size) of 10～100ng.With PC4 albumen on the lattice array interactional target protein takes place and can utilize specific antibody (antibody of for example anti-transcription factor TFIIA, Gal4, Sp1) to detect, as a result Fig. 4 C, Fig. 4 D, Fig. 4 E.

If detect the interaction of albumen and DNA, utilize the autoradiographic technique monitoring mark to have ³²The dna probe of P-dCTP and bound DNA.The result is shown in Fig. 4 F.

As seen from Figure 4, the phenylalanine in the 77th site is absolutely necessary in conjunction with ssDNA for PC4.In addition, the interaction of F77P saltant PC4 and transcription factor TFIIA and Sp1 significantly strengthens.

In sum, the PC4 that single amino acids sudden change (F77P) has taken place except the 77th site has lost ability and the transcriptional activity in conjunction with DNA, the The above results proof also greatly reduces in the proteic activity of PC4 that dual sudden change (K23I/K29A and K35I/K41A) and triple mutant (R27A/K28I/K29A) take place N-end group local area, therefore PC4 mutant: F77P, K23I/K29A, K35I/K41A, R27A/K28I/K29A can be used as cancer therapy drug, reach the effect that treatment depends on the cancer of PC4 by competing with endogenous wild type PC4.

Embodiment 2

Short polypeptide with the amino acid sequence homologous in the activation structure territory of PC4

The inventor designs and has synthesized two peptide species, their sequence is consistent with two PC4 activation structure domain amino acid sequences respectively, proved that by above-mentioned protein arrays analysis (test experience interacts between the protein-protein) and in vitro transcription test experience this two peptide species can combine other with wild type PC4 competition and activate son through the inventor, thereby reached the active effect of PC4 that suppresses.

PC4-AD15, sequence KRKKQVAPEKPVKKQ sees SEQ ID No.1;

And the short polypeptide that is connected with the migration domain accordingly, as TLD-AD15, sequence: PLSSIFSRIGDPKRKKQVAPEKPVKKQ sees SEQ ID No.2;

PC4-S15, sequence D SDSEVDKKLKRKKQ sees SEQ ID No.3;

And the short polypeptide that is connected with the migration domain accordingly, as TLD-S15, sequence: PLSSIFSRIGDPDSDSEVDKKLKRKKQ sees SEQ ID No.4.

Embodiment 3

With the bonded simulation activation structure of PC4 domain polypeptide

(sequence is the amphipathic helix polypeptide: ELQELQELQALLQQQ, see SEQ ID No.5) can simulate a kind of activation structure territory and PC4 generation strong interaction, sealed the activation structure territory of PC4, suppressed PC4 and endogenous activation and interact, thereby suppress the PC4 activity;

And the short polypeptide that is connected with the migration domain accordingly, as TLD-AH15, sequence: PLSSIFSRIGDPELQELQELQALLQQQ sees SEQ ID No.6.

Embodiment 4

Utilization suppresses the chemical compound of PC4 protein active based on the high flux screening detection system screening of FRET (fluorescence resonance energy transfer) technology.

Screen the chemical compound of inhibition PC4 protein active of the present invention and use amphipathic helix (AH) the domain polypeptide that is marked with fluorescence as energy donor, the PC4 albumen of mark fluorescent is as energy acceptor.In case PC4 and amphipathic helix domain polypeptide form complex, promptly owing to the interaction force between two molecules makes that distance is very near (apart from about 1～10nm) between donor and the receptor, what observed this moment mainly is the emission light of PC4, because intermolecular fluorescence resonance energy is transferred to PC4 albumen from amphipathic helix domain polypeptide.The exciting light of amphipathic helix domain polypeptide and the emission light of PC4 are subjected to the monitoring that fluorescence intensity is read the plate instrument.The effect of the inhibition PC4 protein active of any chemical compound can both be measured by the variation of monitoring the fluorescence intensity that is read.General screening two compounds: 1) micromolecule synthetic; 2) native compound.Certainly, these chemical compounds also need further be confirmed, prove that it can become candidate's anticarcinogen that a kind of potential treatment depends on the malignant tumor of PC4.

The micromolecule synthetic comprises commercial the developing with some scientific research institution inside of obtaining of synthesizing.Selected chemical compound is kept in 96 orifice plates or 384 orifice plates, and sets up their a detailed data storehouse.Each hole on the Sptting plate all has 10～50ul to comprise chemical compound to be detected (the 384 orifice plate 10ul that concentration is 1mM, 96 orifice plate 50ul) reactant mixture, and also comprised PC4 albumen and the amphipathic helix domain polypeptide that is marked with fluorescence in the reactant mixture.4 positive control holes (with the concentration fluorescence that is the unlabelled amphipathic helix domain of 10mM polypeptide surrogate markers) and 4 negative controls (water substitutes fluorescently-labeled amphipathic helix domain polypeptide as energy donor) are all arranged on each plate.Read the optical density value that plate instrument (Molecular Dynamics company produces) reads OD535 with fluorescence intensity.The anticancer effect of the positive molecule that screens need adopt different detection methods further to confirm, described different detection method comprises that external protein-protein interaction detects, transcribes and detect and the preceding drug effect detection of adopting cell model and animal model to carry out of clinical trial.Most prescription drugs all is a native compound, and anticarcinogen more than 50% and the anti-infective that can buy on the market all come from natural product.

For identifying that can native compound suppress the proteic activity of PC4, can choose the chemical compound that some derive from soil microorganism, Marine microorganism, plant or other nature materials, utilize high throughput system that their screenings are detected based on the FRET (fluorescence resonance energy transfer) technology.The raw material that screens can be further purified again, identify and further confirm in some above-mentioned different detection systems again.

Above-mentioned summary of the invention and embodiment only for illustrating inventive concept of the present invention and main points, can not limit protection scope of the present invention with this.All equivalences of doing according to embodiments of the invention and in conjunction with spirit of the present invention change or modify, and these all are that those skilled in the art can expect, all should be encompassed in protection scope of the present invention.Moreover, relevant position mark in the text all such as the teaching material of all references or other document publications here.

List of references

1.Ge，H.and?Roeder，R.G.(1994)Purification，cloning?and?characterization?of?a?human?coactivator，PC4，that?mediates?transcriptional?activation?of?class?II?genes.Cell?78，513-523.

2.Kretzschmar，M.et?al.，(1994)A?novel?mediator?of?class?11?gene?transcription?with?homology?to?viral?immediate-early?transcriptional?regulators.Cell?78，525-534.

3.Kishore，A.H.et?al.，(2007)p53?regulates?its?own?activator-transcriptional?coactivator?PC4：a?p53?responsive?gene.Biochem.J.BJ20070390.

4.Banerjee，S.et?al.，(2004)General?transcription?coactivator?PC4?activates?p53?function.Mol.Cell.Biol.24，2052-2062.

5.Ge，H.，Zhao，Y，Chait，B.T.and?Roeder，R.G.(1994)Phosphorylation?negatively?regulates?the?function?of?coactivator?PC4.Proc.Natl.Acad.Sci.USA?91，1269112695.

6.Kumor，P.B.R.et?al.，(2001)p300-mediated?acetyl?ation?ofhuman?transcriptional?coactivator?PC4?is?inhibited?by?phosphorylation.J.Biol.Chem.276，16804-16809.

7.Pilch，B.et?al.，(2001)Specitlc?inhibition?of?serine-and?arginine-rich?splicing?factors?phosphorylation，spliceosome?assembly，and?splicing?by?the?antitumor?drug?NB-506.Cancer?Resaerch?61，6876-6884.

8.Chen，A.Y?et?al.，(2005)Silatecan?OB-67?is?a?novel?DNA?topoisomerase?ltargeted?radiation sensitizer.Mol.Cancer?Thera.4，317-324.

9.Das，C.et?al.，(2006)Transcripional?coactivator?PC4，a?chromatin-associated?protein，induces?chromatin?condensation.Mol.Cell.Biol.26?8303-8315.

10.Kleivi，K.et?al.，(2007)Gene?expression?profiles?of?primary?colorectal?carcinomas，liver?metastases，and?carcinomatoses.Mol.Cancer?6，1-16.

11.Holt?et?al.，(2003)Trends?in?Biotechnology?21：484-490.

12.Harlow?and?Lane，(1988)Antibodies：A?Laboratory?Manual.Cold?Spring?Harbor?Laboratory?Press，Cold?Spring?Harbor，N.

13.Morrison?et?al.，(1984)Proc.Natl.Acad.Sci.USA，81：6851-6855.

14.Neuberger?et?al.，(1984)Nature?312：604-608.

15.Takeda?et?al.，(1985)Nature?314：452-454.

16.Hunter?et?al.，(1975)J.Biol.Chem.250：409-17.

17.Manche?et?al.，(1992)Mol.Cell.Biol.12：5239-48.

18.Minks?et?al.，(1979)J.Biol.Chem.254：10180-3.

19.Elbashir?et?al.，(2001)Nature?411：494-8.

20.Bass，(2001)Nature?411：428-9.

21.Elbashir?et?al.，(2001)Genes?Dev.15：188-200.

22.Jaeger?et?al.，(1989)Proc.Natl.Acad.Sci.USA?86：7706.

23.Turner?et?al.，(1988)Annu.Rev.Biophys.Biophys.Chem.17：167.

24.Kehlenback?et?al.，(1998)J.Cell?Biol.141：863-74.

25.Ge，H.(2000)UPA，a?universal?protein?array?system?for?quantitative?detection?of?protein-protein，protein-DNA，protein-RNA?and?protein-ligand?interactions.Nucl.Acids，Res.28，e3.

26.Ge，H.et?al.，(1998)Isolation?of?cDNAs?encoding?novel?transcription?coactivator?p52?and?p75?reveals?an?alternate?regulatory?mechanism?of?transcriptional?activation.EMBO?J.17，6723-6729.

27.Oess?et?al.，(2000)Novel?cell?permeable?motif?derived?from?the?PreS2-domain?of?herpatitis-B?virus?surface?antigens.Gene?Ther.7，750-758.

28.Stoeckl?et?al.，(2006)Identification?of?a?structural?motif?crucial?for?infectivity?of?herpatitis?B?viruses.Proc.Natl.Acad.Sci.USA?103，6730-6734。

Claims (1)

1. one kind is used for the treatment of or the medicine of prophylaxis of cancer, described medicine has comprised the PC4 antagonist of effective dose, it is characterized in that: described PC4 antagonist is monoclonal anti PC4 antibody or the polyclonal antibody that contains described monoclonal anti PC4 antibody, described monoclonal anti PC4 antibody is the subtype specificity antibody of PC4, and is chimeric antibody.

Images