CN1399680A - Epithelial cell growth inhibitors - Google Patents

Abstract

Epithelial cell growth inhibitors differentially express in normal and cancerous epithelial cells. The ECGI proteins and nucleic acid sequence encoding them are useful in the diagnosis and treatment of epithelial cell cancers, for example prostate, ovarian, colon cancer, and the like.

Description

Epithelial cell growth inhibitors

Invention field

The present invention relates to be used to diagnose and treat the epithelial cell growth inhibitors family of cell carcinoma.

Background of invention

Cell carcinoma, for example prostate cancer, mastocarcinoma, colorectal carcinoma, lung cancer, pancreas cancer, ovarian cancer, spleen cancer, carcinoma of testis, thymic carcinoma etc. are to be the disease of feature with the unusual accelerating growth of epithelial cell.This accelerating growth causes that at first tumour forms.At last, tumour also can be transferred to different organ sites.Although getting along with aspect the diagnosis of various cancers and the treatment, these diseases still cause significant mortality ratio.

Early detection can strengthen treatment for cancer greatly.Yet the early detection disease has difficulties.For example, the difficult touch of organ (as prostate gland, ovary and other organ) of containing epithelium.Do not have frequent screening and suitable marker, be difficult to detect tumor growth unusual in these organs.The ratio that the very big shortcoming of existing cancer diagnosis test is false positive and false negative result is very high, thereby makes the reliability of existing test less than desired.For this reason, be starved of diagnosis and the treatment of identifying that the diagnostic reagent make new advances and new therapeutical agent improve cancer (prostate cancer, mastocarcinoma, colorectal carcinoma, lung cancer, pancreas cancer, ovarian cancer, spleen cancer, carcinoma of testis, thymic carcinoma etc.).

A kind of new specificity mammary cell growth inhibitor breast statin (Mammastatin) has obtained recently identifying, and has made specificity analysis.This breast statin can be by following each clonal expression: MammA (PCT/US97/18026, ATCC#97451, preservation on February 22 in 1996); MammB (PCT/US97/27147, ATCC# PTA-2091, preservation on June 15 in 2000); And Mammc, at the PCT application PCT/US00/ that awaits the reply that submits on the same day with the application 16900In (ATCC# is described to some extent PTA-2090, preservation on June 15 in 2000).

The breast statin is produced and secretion by normal mammary cell, can detect in the blood sample of normal individual.In the mastocarcinoma patient, the blood concentration of this mammary cell growth inhibitor (specifically being the active phosphorylation form of breast statin) reduces or does not have.The albumen that contains active breast statin (by the emiocytosis of normal people's mastocarcinoma) can reduce tumour size and number effectively, and can stop the tumor growth of patient with advanced cancer.

The epithelial cell growth inhibitors similar with the breast statin is found, separates and CHARACTERISTICS IDENTIFICATION.These inhibitor suppress to have the partial sequence homogeny at 5 of sequence ' end and breast, and have seldom or do not have homogeny at 3 of molecule ' end.The same with the breast statin, the variant expression in the normal epithelium cell tissue of newfound epithelial cell growth inhibitors (ECGI) family, but in cancer epithelial cell tissue, do not express.In addition, the same with the breast statin, newfound epithelial cell growth inhibitors family can be detected in the blood sample of normal individual, but can not detect (shown in hereinafter embodiment) in the cell carcinoma blood samples of patients.

Summary of the invention

In many different epithelial cells, identified at present epithelial cell growth inhibitors (ECGI) family.These ECGI are differential expression in normal epithelium cell, but do not express in epithelial cancer cells.Shown in hereinafter embodiment, in various epithelial cell tissues (comprising prostate gland, colon, ovary, lung, spleen, testis, thymus gland and other epithelial cell tissue), found breast statin sample ECGI albumen.

ECGI of the present invention expresses in normal epithelium cell, but does not express in the cancer epithelial cell.Make the nucleic acid array hybridizing of proteic nucleotide sequence of coding breast statin sample ECGI and coding breast statin.ECGI albumen also can with anti-breast statin antibodies.The aminoacid sequence (PRT-6, SEQ ID NO:4) of coding ECGI is separated and made CHARACTERISTICS IDENTIFICATION (PRT-6, ATCC# in the prostatic cell PTA-2092, preservation on June 15 in 2000), this describes among embodiment hereinafter to some extent.

Because ECGI of the present invention differential expression and not expressing in the cancer epithelial cell in normal epithelium cell, but therefore existence or its content of analysing ECG I come diagnosing cancer and/or monitoring therapeuticing effect with this.The ECGI albumen of the present invention and this proteic nucleic acid of encoding also provide the useful therapeutical agent that suppresses epithelial cell growth, prevents tumour formation and treatment cancer.

The accompanying drawing summary

Figure 1A is the mRNA test panel synoptic diagram that shows the specific tissue mRNA position of being analyzed.

Figure 1B is the computer scanning image of Northern trace, and it has shown the mRNA hybridization of the various tissues of scheme shown in breast statin nucleotide sequence and Figure 1A.

Fig. 2 is the computer scanning figure of Dot blot test, and it has shown with condition (conditioned) nutrient solution of anti-breast statin antibody 7G6 detection contrast, breast statin standard protein, mastocarcinoma patient serum sample, normal and carcinous human prostate cell (result).

Fig. 3 is the computer scanning image of Western trace test, and it has shown the result who detects normal people's mammary cell lysate (A), human prostata cancer LnCap cell pyrolysis liquid (B), MCF7 mastocarcinoma cell pyrolysis liquid (C) and normal human prostate cell pyrolysis liquid (D) with anti-breast statin antibody 7G6.

Fig. 4 is the computer scanning image of Western trace test, and it has shown the result who detects the cell pyrolysis liquid of normal prostatic cell (A), LnCap prostate cancer cell (B), normal colon cell (C) and colon cancer cell (D) with anti-breast statin antibody 7G6.

Fig. 5 is the computer scanning figure of Western trace test, and it has shown the result who detects the cell pyrolysis liquid of Proliferation of Human Ovarian Cell (B), normal people's gonad cell (C) and normal people's mammary cell (D) with anti-breast statin antibody 7G6.Swimming lane A is the molecular weight standard product.

Fig. 6 is the computer scanning image of Dot blot test, it shown with anti-breast statin antibody 7G6 detection NAM (A, C, D) and the result of the serum sample of patients with prostate cancer (B).

Fig. 7 is the calculating scanning image that contains the dna gel of the prostate gland ECGF dna clone of inferring.

Fig. 8 be prostate gland ECGI chart signal and with the structural relation of other sequence.

Detailed Description Of The Invention

Albumen of the present invention:

" epithelial cell growth inhibitors (ECGI) albumen " of the present invention is defined as the breast inhibin sample albumen with its growth activity of inhibition that is produced by normal epithelium cell in this article. Active inhibition ECGI albumen of the present invention is less or do not have in the cancer epithelial cell. It seems that ECGI protein family disclosed herein comprise that each epithelial tissue is had specificity and to types of organization not or the very little active inhibitor that suppresses arranged. As hereinafter the institute in greater detail, estimate that every kind of ECGI albumen contains growth inhibition domain and tissue specificity domain.

ECGI albumen of the present invention shows and the remarkable homology of breast inhibin, and the latter is a kind of mammary cell growth inhibitor that is produced by normal person's mammary cell, and it has been proved and has can be used for diagnosis and treatment mastocarcinoma (PCT/US97/18026). ECGI albumen can with one or more anti-breast inhibin antibody such as 7G6 (Neomarkers, Freemont, CA) combination, its nucleotide sequence of nucleotide sequence and coding breast inhibin of encoding has remarkable homology.

That hereinafter reports among the embodiment studies confirm that ECGI albumen differential expression in the normal epithelium cell tissue, but does not express in cancer epithelial cell tissue (comprising breast, prostate gland, ovary and colon).The same with the breast statin, it seems that ECGI albumen of the present invention be two or three bands in Western trace for example: a main band and one or two less not too significant band.Verified, this expression pattern of breast statin is because this proteinic phosphorylation.When in two place phosphorylations, the molecular weight of breast statin is approximately 53 kilodaltons.Less breast statin (49 and 44 kilodalton) is corresponding to a position or do not have the position by phosphorylation.The proteic phosphorylation of breast statin is relevant with its inhibition activity.

The about size that has shown the ECGI that various epithelial cell tissues produce with the Western trace of anti-breast statin antibody 7G6 detection ECGI.As the demonstration (for example referring to Fig. 4-5) that hereinafter embodiment is more complete, the ECGI of prostatic cell moves to about 55 kilodalton places in the Western trace, not too significant, less band points out its phosphorylation form similar to breast statin finding at 51 and 46 kilodalton places.The ECGI of colon cell migrates to about 50KD place, and not too significant band is about 47 and the 43KD place.The ECGI of gonad cell migrates to about 60KD place.

The nucleotide sequence of coding ECGI

Nucleotide sequence of the present invention is defined as the proteic nucleotide sequence of ECGI of the above-mentioned definition of coding in this article.Coding ECGI proteic nucleotide sequence has significant sequence homology with the nucleotide sequence of coding breast statin, and they can be under highly rigorous condition and the nucleic acid array hybridizing of coding breast statin.

Breast statin sample epithelial cell growth inhibitors preferably has basically homogeny (have 90% at least, preferable have at least 95% identical) in about 1000 Nucleotide that adjoin of the nucleotide sequence of coding breast statin.The nucleic acid of coding breast statin comprises MammA (PCT/US97/18026, ATCC#97451, preservation on February 22 in 1996); MammB (PCT/US97/27147, ATCC# PTA-2091, preservation on June 15 in 2000); With MammC (ATCC# described herein PTA-2090, preservation on June 15 in 2000) the DNA inset.The measured consensus sequence of known breast statin clone is presented at hereinafter sequence contrast table 5 and SEQ IDNO:1 (MammA); SEQ ID NO:2 (MammB); Among the SEQ ID NO:3 (MammC).Prostate gland ECGI nucleotide sequence (SEQ ID NO:4) is presented in the table 1,2 and 5.

ECGI can obtain from specificity epithelial cell nucleic acid amplified library, for example adopts inner breast statin primer and/or hybridizes with the breast statin under rigorous condition.As institute hereinafter in greater detail, shown with the nucleotide sequence of breast statin hybridization to be present in (see figure 1) in many epithelium (comprising central nervous system, heart, small intestine, large intestine, appendix, rectum, lymphocyte, medullary cell, lung and air flue, bladder, uterus, prostate gland, testis, ovary, liver, pancreas, suprarenal gland, sialisterium and mammary gland).

Nucleotide sequence and the breast statin of isolated ECGI have the homogeny more than 95% at molecule 5 ' end from prostatic cell for example, yet, there is seldom or do not have the sequence homogeny at 3 ' end.Estimate to suppress to have the growth-inhibiting structural domain that 5 of homogeny ' end contains this molecule, and contain the tissue specificity structural domain with breast statin homogeny 3 ' end seldom with breast.

Diagnostic method

The present invention also provides the in vitro tests of active inhibition ECGI in a kind of detection patient's sample (comprising tissue, cell and body fluid).By proteic existence of ECGI and type in patient's sample are associated with normal or carcinous human epithelial cell, diagnosable cell carcinoma and late period metastatic disease.Analyze the ECGI albumen in blood samples of patients or the tissue sample, for example the proteic abundance of analysing ECG I and/or its molecular weight form.As described below, lacking or losing with the specificity epithelial cell that shows the metastatic disease in late period of the phosphorylation form of ECGI, especially higher molecular weight is associated.

With various known analysis tools and method (comprising immunity test, hybridization, round pcr etc.) but analysing ECG I.Preferred methods is to analyze patient's sample with anti-ECGI antibody by immunity test (comprising ELISA, Western trace and Dot blot).The preferred reorganization ECGI standard substance that adopt provide typical curve, with the level of detection by quantitative inhibitor reliably.Among the embodiment, enumerated Dot blot test and the test of Western trace typical case hereinafter as these immunity tests.In another preferable embodiment of the present invention, express analysis bank tissue samples (as the tumor biopsy thing) with immunohistochemical method or by the ECGI that cultivates the patient tumors cell and detect culture.

In a good especially embodiment, the test of diagnosis cell carcinoma comprises at least two strain specific antibodies: a kind ofly identify that sampling is organized as the antibody of epithelium, as anti-cell keratin antibody and a kind of anti-ECGI specific antibody.For example, utilize the immunoblotting form, suspection is contained the prostata tissue homogenate of prostate cancer cell, on the SDS/PAGE gel, separate, transfer on the film, with antikeratin antibody and anti-prostate gland ECGI antibody test.Detect bonded antibody with resisting with appropriate flags system (as peroxidase or alkaline phosphatase) link coupled isotype specificity two.Make the film development that contains bonded first and second antibody with known colorimetric technology or fluorescence technique then, and use the currently known methods quantitative assay.

In the embodiment of the best, come size and/or the phosphorylation form of ECGI in the analytic sample by for example Western trace with anti-ECGI antibody.The minimizing or the disappearance of high molecular ECGI albumen form are associated with terminal cancer.

Diagnostic kit of the present invention comprises the nucleotide sequence (for example in contrast) of ECGI albumen or coding ECGI.This diagnostic kit randomly contains can be with to be detected or treat one or more antibody of epithelial cell ECGI bonded of quantitative assay.Antibody capable maybe can be tissue-specific ECGI antibody in conjunction with breast statin spline structure territory (for example 7G6).Perhaps, diagnostic kit comprises one or more amplimers or the hybridization probe (for example being used for the hereinafter probe of embodiment) that is used for increasing and/or detects the nucleotide sequence of coding epithelial cell ECGI.

Therepic use

The ECGI albumen that is used for the treatment of purposes is produced under serum-free condition or by recombination method by the epithelial cell culture.Preferable, ECGI albumen produces in yeast or more high eukaryotic cell, to realize this proteic phosphorylation.Recombinant protein is produced by host cell or produces by synthetic method.

Functional ECGI can come administration by known phosphorprotein medication, and preferably drug administration by injection to improve the inhibitor level in the blood, increases the interaction of inhibitor and required epithelium.

The known method of this albumen phosphoric acid pharmaceutical grade protein transportation art flows to the patient.Usually, with this inhibitor and conveying mixed with excipients, drug administration by injection then.

The dosage of inhibitor can be determined that it is different because of the type and the disease severity of treatment by those skilled in the art.Because the breast statin can suppress about 50% breast growth of cancer cells external with 10 nanogram(ng)s/ml concn, with 20-25 nanogram(ng)/ml concn the breast growth of cancer cells is stopped, so the useful therapeutic dose scope of ECGI is to give about 2.5 microgram to 250 micrograms every day.Preferably give the dosage of about 125 micrograms every day.The purpose of administration is to make final body dosage in physiological range (for example 15-50 millimicro grams per milliliter) or high slightly scope (for example 25-75 millimicro grams per milliliter).For clinical use, preferable dosage range is that the initial treatment to metastatic disease is about 500 millimicro grams per milliliters, and maintenance dose is about 50 millimicro grams per milliliters then.In the clinical study of adopting the breast statin, every day, dosage was about the alleviation that 50-750 millimicro grams per milliliter just is enough to induce IV phase mastocarcinoma patient.

Because active ECGI is phosphorylated protein, need to estimate this inhibitor of multiple doses, with the ECGI of the growth-inhibiting level of keeping ECGI in the blood samples of patients.In addition, because ECGI plays cytostatics effect rather than cytocide effect usually, estimate that therefore the maximum effect of this inhibitor need often keep cell carcinoma patient's inhibitor level.

In this preferable application, ECGI gives with high dosage (greater than 50 millimicro grams per milliliters, the preferable 50-500 millimicro grams per milliliter that is about), with the induced tumor regression.Lower maintenance dose (less than 50 millimicro grams per milliliters, preferable is 20-50 millimicro grams per milliliter) is used for preventing growth of cancer cells.

The useful dosage of keeping breast statin physiological level in the blood has been pointed out in the clinical experiment that the breast statin is given IV phase mastocarcinoma patient.Administration should be carried out every day, but can be for example to infuse continuously by the slow release drop, or every 2-3 day injects once.The evidence prompting, successive administration may be induced feedback inhibition.Therefore, preferable dosage regimen is the breast statin that gives per daily dose, continues about 25-28 days, not administration in 2-5 days then.

Diagnostic test

The existence whether test that the present invention detects the inhibitor that works in people's tissue and the serum can be used to screening cell carcinoma patient, screening has the crowd of the highly dangerous that develops into cell carcinoma, detect epitheliomatous early onset thereof, and the inhibitor level of during treating, monitoring the patient.For example, the blood ECGI that analyzes the patient may show, compares with the prostate gland ECGI distribution situation before normal control or the patient, and the content of the prostate gland ECGI of high-molecular weight phosphorylation reduces.The danger of this variation and prostate cancer increases, the early onset thereof of prostate cancer and late period metastatic prostate cancer relevant.The diagnostic test of the prostate gland ECGI with active 55kD of phosphorylation should be Western trace immunity test or the ELISA that adopts the anti-ECGI antibody of specificity.For example, the serum screening should use immunity test (as ELISA, Western trace or Dot blot test) to carry out.

For optimal results, patient's sample should be after sampling in the short period of time (in 1 week) test, be preserved under 4 ℃ and (be less than 1 year), or the long-term frozen preservation.Best is sample to be refrigerated to test.

Embodiment

Can understand the present invention better with reference to the following example, but the present invention also is confined to these embodiment never in any form.

Embodiment 1

Many tissue expressions of ECGI

(Clonetech carries out the Northern engram analysis on Inc.#7775-1), to confirm the expression of ECGI in various epithelial cell tissues at many tissue expressions array.With the breast statin EcoR1 fragment of the digoxigenin labeled of about 1800 base pairs that contain pMammC3 ' zone (SEQID NO:3, approximately Nucleotide 359-end) as probe.The breast statin cDNA of DIG mark was hybridized 16 hours for 65 ℃ with this array in 10 milliliters of easy HYB solution (Roche), and 65 ℃ of washings according to manufacturer's specification sheets, with anti-DIG antibody hybridization, are developed with CSPD.Then with trace room temperature exposure Kodak X-OMAT film 30 minutes.

The tissue plane figure of many tissue expressions array is presented among Figure 1A.The mRNA hybridization of breast statin cDNA and this array is presented among Figure 1B, the figure illustrates the kind of the epithelial cell tissue of expressing breast statin sample ECGI sequence.Comprise with the specific tissue of breast statin cDNA hybridization: central nervous system, heart, small intestine, large intestine, appendix, rectum, lymphocyte, medullary cell, lung and air flue, bladder, uterus, prostate gland, testis, ovary, liver, pancreas, suprarenal gland, sialisterium and mammary gland.

Embodiment 3

Normal prostatic cell and carcinous prostatic cell

The carcinous prostatic cell LnCap that cultivates the surgery normal prostatic cell of sample gained and obtain from American type culture collection (ACTT) analyzes the generation of prostate gland ECGI.Cell is containing 40 μ M calcium, is adding in the DMEM/F12 nutrient solution of the horse serum that 5%Chelex handled, 10 millimicro grams per milliliter EGF, 10 mcg/ml Regular Insulin, 100 millimicro grams per milliliter Toxins,exo-, choleras and 1 mcg/ml hydrocortisone and cultivated 4 days.Collection condition nutrient solution sample and analysis then.

Normal people's mammary cell that patient's sample is obtained is cultivated in same medium, adopts the breast statin that is secreted in the nutrient solution in contrast.The serum of analyzing mastocarcinoma patient acquisition in addition is also with comparing.

Collect liquid sample, be loaded on the nitrocellulose membrane on the Dot blot device by suction.Detect this film with anti-breast statin antibody 7G6 then, with the goat anti-mouse antibody detection antibodies of alkali phosphatase enzyme mark.Develop the color with NBT/BCIP substrate system (Life Technologies).The result is presented among Fig. 2.

Anti-breast statin antibody capable identification normal prostatic cell produces and a kind of protein that carcinous prostatic cell can not produce.This is similar to this antibody capable identification mammary cell growth inhibitor breast statin that the mastocarcinoma cell can not produce by normal mammary cell generation.These data are the data in 1 in conjunction with the embodiments, prove in other epithelial cell tissue, especially produced breast statin sample ECGI in the prostatic cell.

Embodiment 3

The differential expression prostate gland of ECGI in prostate gland, colon and ovary

As described in above-mentioned embodiment 2, cultivate the normal prostatic cell (Clonetech, Inc.), LnCap prostate cancer cell (ATCC), MCF7 mastocarcinoma cell (ATCC) and normal people's mammary cell (from hospital's tissue acquisition).Cultivate after at least 48 hours, make cell cracking in sample loading buffer, as probe, use the existence of Western engram analysis ECGI with anti-breast statin antibody 7G6.Contrast (A) with normal people's mammary cell albumen (NHMC) lysate (1 mg/ml) as the breast statin.Data presentation is in Fig. 3.

Normal prostatic cell pyrolysis liquid (D) contains can be by the protein of anti-breast statin antibody recognition, and prostate cancer cell (LnCap) (B) and (C) this albumen not of mastocarcinoma cell (MCF7).Its size of the protein of identification is similar to breast statin (A) in the prostatic cell lysate (D).Colon and prostate gland

As described in above-mentioned embodiment 2, cultivate the normal prostatic cell (Clonetech, Inc.), LnCap prostate cancer cell (ATCC), Sw 948 colon cancer cells (ATCC) and normal colon epithelial cell (from the acquisition of patient's surgical operation tissue).In sample loading buffer, prepare cell pyrolysis liquid, as probe, use the expression of Western engram analysis ECGI with anti-breast statin antibody 7G6.

As shown in Figure 4, normal prostatic (A) and normal colon (C) epithelial cell have been expressed can be by a kind of protein of anti-breast statin antibody recognition, and the cancer cells of these tissues (B D) can not express this protein.Shown similar of breast statin in proteinic differential expression and the breast tissue.In addition, normal prostatic is similar to the being seen phosphorylation pattern of breast statin that normal people's mammary cell produces with the strip pattern that the Western of colon trace shows.Show a bigger main band and two less fuzzy bands among the figure.This pattern is relevant with the phosphorylation of breast statin.

Western engram analysis (Fig. 4) demonstrates the molecular weight that prostate gland ECGI has about 51 kilodaltons; Colon ECGI shows the molecular weight with about 50 kilodaltons.Ovary

As described in above embodiment 2, cultivate OvCar-ovarian cancer cell (ATCC), normal people's gonad cell (patient's surgical operation tissue) and normal people's mammary cell (patient's surgical operation tissue).Cultivate after at least 48 hours, remove growth medium, clean cell until thickness, make direct lysate with salt solution and SDS-PAGE sample loading buffer.Collect lysate, separate on 10%SDS-PAGE, electrophoretic transfer is to nitrocellulose membrane, with the antibody test of the anti-breast statin of 7G6.Data presentation is in Fig. 5, and wherein swimming lane A contains the molecular weight standard product; B is an OvCar ovarian cancer cell lysate; C is normal people's gonad cell lysate; D is normal people's mammary cell lysate.

Fig. 5 proves that this breast statin sample ECGI albumen is produced by normal people's ovary tissue, and by anti-breast statin antibody recognition.This albumen is not expressed in the ovarian cancer cell of being analyzed.The molecular weight of ovary ECGI is approximately 60 kilodaltons.

Embodiment 4

The otherness of prostate gland ECGI detects in the blood

Analyzed the existence of prostate gland ECGI in the serum sample of three healthy male volunteers, and with the serum of itself and patients with prostate cancer relatively.Serum sample is with 400 microlitres and 200 microlitre sample pipetting volumes, and is duplicate.Utilize vacuum that sample is drawn onto on the nitrocellulose membrane in the 96 hole Dot blot devices.Detect filter membrane with anti-breast statin antibody 7G6 then, with the colour developing of NBT/BCIP substrate.Data presentation is in Fig. 6.

The standard substance conditioned medium that normal people's mammary cell (NHMC) culture produces is used for comparison.Bipartite standard substance contain 400,200,100,50,25,12 and 6 microlitre NHCM nutrient solutions.To NAM (A, C, D) and the serum sample of prostate cancer adult patients (B) test, adopt 400 and 200 microlitre serum samples.(A, C D) have tangible signal indicating to have prostate gland ECGI to normal serum, and the serum of patients with prostate cancer shows to have only faint signal.

Embodiment 5

The inhibition activity of prostate gland ECGI

With the normal prostatic cell (Clonetech, Inc.), PC3 and LnCap prostate cancer cell (ATCC) be with every milliliter 5.0 * 10 ⁴The density of cell is inoculated in 12 orifice plates of the RPMI substratum that contains 10% foetal calf serum.After 24 hours, in culture, add 10% conditioned medium.Each sample carries out in triplicate.Dull and stereotyped in 37 ℃, 5%CO ₂Under cultivated 6 days, cultivate the end of term, (Cetrimide) makes lysis with Cetrimide, uses the Colter rolling counters forward.More treated and undressed hole are calculated and are suppressed percentage ratio, and data presentation is in following table.The PC3 cell of androgen insensitivity is not suppressed by the conditioned medium of normal prostatic cell culture fluid or normal prostatic cell gained.On the contrary, the grown cultures liquid that the LnCap cell is added into suppresses, the nutrient solution that the inhibition degree of the nutrient solution that normal prostatic obtains obtains greater than cancer cells to a certain extent.

Cell type	The inhibition % of normal prostatic nutrient solution	The inhibition % of tumor of prostate nutrient solution
			????LnCap#1	????22.5+/-3.3	??8.3+/-0.4
????LnCap#2	????22.7+/-0.6	??16.7+/-15.8
			????PC3	????0	??0

Embodiment 6

The separation of prostate gland ECGIDNA and evaluation

Produce nucleic acid library from the mRNA of normal prostatic cell (patient's surgical operation tissue) and LnCap prostate tumor cells (ATCC).

The nucleotide sequence in normal prostatic cell and prostate cancer cell library is inserted in the carrier, be used for transform bacteria.As mentioned above, by under rigorous condition with the breast statin nucleic acid probe hybridization of digoxigenin labeled, filter out the bacterial colony that can express normal and carcinous prostatic cell nucleotide sequence.

Select positive bacterium colony, in LB meat soup, grow.The plasmid that purifying obtains from positive bacterium colony is with ECO R1 and Xho1 digestion, to discharge the cDNA inset.Go up the DNA that separates through digestion at 1% sepharose (seeing Fig. 7 A) then, the breast statin fragment of using digoxigenin labeled is carried out the Southern engram analysis as probe to separated DNA.As shown in Figure 7, isolate two prostate gland ECGI clones, each clone has the size of about 2kb; A clone separates from normal prostate tissue library (PRN2.1) and obtains, and one is separated from LnCap prostate tumor cells library (PRT-6) and to obtain.

PRT-6 is made further CHARACTERISTICS IDENTIFICATION, measure its nucleotide sequence.As shown in table 1 below, the nucleotide sequence of coding prostate gland ECGI is in molecule 5 ' end and breast statin basic identical (greater than 90%), and 3 of molecule ' end have seldom or do not have a homogeny.The different zone of these phase Sihes with pictorialization in Fig. 8.

Embodiment 7

Separation and the CHARACTERISTICS IDENTIFICATION of prostate gland ECGI DNA

Make up nucleic acid library from the mRNA of normal prostatic cell (obtaining) and LnCap prostate tumor cells (ATCC) from patient's surgical operation tissue.CDNA comes transformed into escherichia coli with the library, and colony hybridization is carried out in inoculation.Adopt with following exterior PC R primer M200 and M2200

[SEQ?ID?NO：5]M200：GCGCCGGCCGGGCGCGACCCG

[SEQ ID NO:6] M2200:GCAATCTCAGCGCACTGCTGC makes the breast statin C fragment of PCR generation digoxigenin labeled and screens bacterium colony.

As mentioned above, the bacterial colony of expressing prostate gland ECGI clone under rigorous hybridization conditions can with the breast statin probe hybridization of mark.

Embodiment 8

The homology of prostate gland ECGI

Nucleotide sequence among the prostate gland ECGI sequence contrast GenBank is analyzed.The part of these two molecules of 28SmRNA and Hip55 demonstrate with prostate gland ECGI sequence in structural domain some similaritys are arranged.

The homology of 28SmRNA is present in (people such as Hu, 1999, PNAS96:1339-1344 in many important growth regulatory gene sequences by evaluation; People such as Mauro, 1997, PNAS 94:422-427).Hip55 be a kind of can with hematopoiesis my late grandfather 1 type kinase (a kind of protein of the src of participation signal transduction passage) bonded protein (people such as Ensena, 1999, J.Biol.Chem.274:33945-50).

Utilize the known open reading frame of Hip55, infer out this prostate gland clone's putative amino acid sequence.As shown in table 3 below, this translation thing comprises several inner codons of ending.

In addition, utilize Hip55 ORF, infer out the putative amino acid sequence of MammB and MammC sequence, shown in table 4 and 5.

Table 1

PMammC and prostate gland ECGI

Table 2

Prostate gland ECGI homology

Table 3

The prostate gland ECGI aminoacid sequence of inferring

H??E??I???P??T??V??P???T??Y??Y???P??A??K???P??Q??1????GCACGAGATT?CCCACTGTCC?CTACCTACTA?TCCAGCGAAA?CCACAGCCAA

CGTGCTCTAA?GGGTGACAGG?GATGGATGAT?AGGTCGCTTT?GGTGTCGGTT

·?E??R??A???W??R??N???Q??R??G??K???K??T??L???L??S?51????GGGAACGGGC?TTGGCGGAAT?CAGCGGGGAA?AGAAGACCCT?GTTGAGCTTG

CCCTTGCCCG?AACCGCCTTA?GTCGCCCCTT?TCTTCTGGGA?CAACTCGAAC

T??L??V??W???H??G??E???E??T??*???E??V??*??N???K??W101????ACTCTAGTCT?GGCACGGTGA?AGAGACATGA?GAGGTGTAGA?ATAAGTGGGA

TGAGATCAGA?CCGTGCCACT?TCTCTGTACT?CTCCACATCT?TATTCACCCT

·A??P??G???A??P??P??V???S??P??R???G??A??R???G??G151????GGCCCCCGGC?GCCCCCCCGG?TGTCCCCGCG?AGGGGCCCGG?GGCGGGGTCC

CCGGGGGCCG?CGGGGGGGCC?ACAGGGGCGC?TCCCCGGGCC?CCGCCCCAGG

·?R??P??C???G??P??P???V??K??Y??H???Y??S??D???R??F201????GCCGGCCCTG?CGGGCCGCCG?GTGAAATACC?ACTACTCTGA?TCGTTTTTTC

CGGCCGGGAC?GCCCGGCGGC?CACTTTATGG?TGATGAGACT?AGCAAAAAAG

T??D??P??V???R??R??G???G??E??P???R??G??A??L???A??S251????ACTGACCCGG?TGAGGCGGGG?GGGCGAGCCC?CGAGGGGCTC?TCGCTTCTGG

TGACTGGGCC?ACTCCGCCCC?CCCGCTCGGG?GCTCCCCGAG?AGCGAAGACC

·A??K??R???P??A??A??R???R??P??G???A??T??R???S??G301????CGCCAAGCGC?CCGGCCGCGC?GCCGGCCGGG?CGCGACCCGC?TCCGGGGACA

GCGGTTCGCG?GGCCGGCGCG?CGGCCGGCCC?GCGCTGGGCG?AGGCCCCTGT

·?A??R??W???G??V??*???L??G??R??Y???T??C??Q???T??V351????GTGCCAGGTG?GGGAGTTTGA?CTGGGGCGGT?ACACCTGTCA?AACGGTAACG

CACGGTCCAC?CCCTCAAACT?GACCCCGCCA?TGTGGACAGT?TTGCCATTGC

Q??V??S??*???G??E??L??R???E??D???R??N??L??P???W??S401????CAGGTGTCCT?AAGGCGAGCT?CAGGGAGGAC?AGAAACCTCC?CGTGGAGCAG

GTCCACAGGA?TTCCGCTCGA?GTCCCTCCTG?TCTTTGGAGG?GCACCTCGTC

·R??A??K???A??R??L??I???L??I??F???S??T??N???T??D451????AAGGGCAAAA?GCTCGCTTGA?TCTTGATTTT?CAGTACGAAT?ACAGACCGTG

TTCCCGTTTT?CGAGCGAACT?AGAACTAAAA?GTCATGCTTA?TGTCTGGCAC

·?S??G??A???S??R??S???F??*??P??F???G??F??*???A??G501????AAAGCGGGGC?CTCACGATCC?TTCTGACCTT?TTGGGTTTTA?AGCAGGAGGT

TTTCGCCCCG?GAGTGCTAGG?AAGACTGGAA?AACCCAAAAT?TCGTCCTCCA

V??R??K??V????T??T??G???I??T??G???L??W??R??P???S??V551????GTCAGAAAAG?TTACCACAGG?GATAACTGGC?TTGTGGCGGC?CAAGCGTTCA

CAGTCTTTTC?AATGGTGTCC?CTATTGACCG?AACACCGCCG?GTTCGCAAGT

·S??D??V???A??F??*??S???F??D??V???G??S??S???Y??H601????TAGCGACGTC?GCTTTTTGAT?CCTTCGATGT?CGGCTCTTCC?TATCATTGTG

ATCGCTGCAG?CGAAAAACTA?GGAAGCTACA?GCCGAGAAGG?ATAGTAACAC

·?A??E??F???T??K??R???W??I??V??H???P??L??I???G??N651????AAGCAGAATT?CACCAAGCGT?TGGATTGTTC?ACCCACTAAT?AGGGAACGTG

TTCGTCTTAA?GTGGTTCGCA?ACCTAACAAG?TGGGTGATTA?TCCCTTGCAC

S??W??D??*???T??V??V???R??Q??V???S??F??T??L???L??M701????AGCTGGGATT?AGACCGTCGT?GAGACAGGTT?AGTTTTACCC?TACTGATGAT

TCGACCCTAA?TCTGGCAGCA?CTCTGTCCAA?TCAAAATGGG?ATGACTACTA

·C??C??C???H??G??N??P???A??Q??Y???E??R??N???R??R751????GTGTTGTTGC?CATGGTAATC?CTGCTCAGTA?CGAGAGGAAC?CGCAGGTTCA

CACAACAACG?GTACCATTAG?GACGAGTCAT?GCTCTCCTTG?GCGTCCAAGT

·?H??L??V???Y??V??L???G??*??G??A???N??G??A???K??L801????GACATTTGGT?GTATGTGCTT?GGCTGAGGAG?CCAATGGGGC?GAAGCTACCA

CTGTAAACCA?CATACACGAA?CCGACTCCTC?GGTTACCCCG?CTTCGATGGT

S??V??G??L???*??L??N???A??S??K???S??E??S??R???P??G851????TCTGTGGGAT?TATGACTGAA?CGCCTCTAAGT?CAGAATCCC?GCCCAGGCGG

AGACACCCTA?ATACTGACTT?GCGGAGATTC?AGTCTTAGGG?CGGGTCCGCC

·T??I??R???Q??R??R??G???A??S??V???G??L??G???*??P901????AACGATACGG?CAGCGCCGCG?GAGCCTCGGT?TGGCCTCGGA?TAGCCGGTCC

TTGCTATGCC?GTCGCGGCGC?CTCGGAGCCA?ACCGGAGCCT?ATCGGCCAGG

·?R??L??S???P??P??A???G??R??P??P???L??H??A???P??R951????CCCGCCTGTC?CCCGCCGGCG?GGCCGCCCCC?CCCTCCACGC?GCCCCGCGCG

GGGCGGACAG?GGGCGGCCGC?CCGGCGGGGG?GGGAGGTGCG?CGGGGCGCGC

R??G??R??A????R??A??P???P??R??A???G??T??G??V???R??C1001???CGCGGGAGGG?CGCGTGCCCC?GCCGCGCGCC?GGGACCGGGG?TCCGGTGCGG

GCGCCCTCCC?GCGCACGGGG?CGGCGCGCGG?CCCTGGCCCC?AGGCCACGCC

·V??P??F???V??L??G??N???G??A??R???P??E??R???R??P1051???AGTGCCCTTC?GTCCTGGGAA?ACGGGGCGCG?GCCGGAAAGG?CGGCCGCCCC

TCACGGGAAG?CAGGACCCTT?TGCCCCGCGC?CGGCCTTTCC?GCCGGCGGGG

·?R??P??S???R??T??A???R??S??W??G???T??W??R??????T1101???CTCGCCCGTC?ACGCACCGCA?CGTTCGTGGG?GAACCTGGCG?CTAAACCACC

GAGCGGGCAG?TGCGTGGCGT?GCAAGCACCC?CTTGGACCGC?GATTTGGTGG

S??I??S??S???P??Q??P???G??K??L???R??S??P??F???L??Q1151???TCCATCTCCA?GTCCTCAGCC?TGGCAAGCTG?AGGAGCCCCT?TCCTGCAGAA

AGGTAGAGGT?CAGGAGTCGG?ACCGTTCGAC?TCCTCGGGGA?AGGACGTCTT

·Q??L??T???Q??P??E??T???H??F??G???R??E??P???A??A1201???GCAGCTCACC?CAACCAGAGA?CCCACTTTGG?CAGAGAGCCA?GCTGCTGCCA

CGTCGAGTGG?GTTGGTCTCT?GGGTGAAACC?GTCTCTCGGT?CGACGACGGT

·?S??R??P???R??A??D???L??P??A??E???E??P??A???P??S1251???TCTCAAGGCC?CAGGGCAGAT?CTCCCTGCTG?AGGAGCCGGC?GCCCAGCACT

AGAGTTCCGG?GTCCCGTCTA?GAGGGACGAC?TCCTCGGCCG?CGGGTCGTGA

P??P??C??L???V??Q??A???E??E??E???A??V??Y??E???E??P1301???CCTCCATGTC?TGGTGCAGGC?AGAAGAGGAG?GCTGTGTATG?AGGAACCTCC

GGAGGTACAG?ACCACGTCCG?TCTTCTCCTC?CGACACATAC?TCCTTGGAGG

·E??Q??E???T??F??Y??E???Q??P??P???L??V??Q???Q??Q1351???AGAGCAGGAG?ACCTTCTACG?AGCAGCCCCC?ACTGGTGCAG?CAGCAAGGTG

TCTCGTCCTC?TGGAAGATGC?TCGTCGGGGG?TGACCACGTC?GTCGTTCCAC

·?G??S??E???H??I??D???H??H??I??Q???G??Q??G???L??S1401???CTGGCTCTGA?GCACATTGAC?CACCACATTC?AGGGCCAGGG?GCTCAGTGGG

GACCGAGACT?CGTGTAACTG?GTGGTGTAAG?TCCCGGTCCC?CGAGTCACCC

Q??G??L??C???A??R??A???L??Y??D???Y??Q??A??A???D??D1451???CAAGGGCTCT?GTGCCCGTGC?CCTGTACGAC?TACCAGGCAG?CCGACGACAC

GTTCCCGAGA?CACGGGCACG?GGACATGCTG?ATGGTCCGTC?GGCTGCTGTG

·E??I??S???F??D??P??E???N??L??I???T??G??I???E??V1501???AGAGATCTCC?TTTGACCCCG?AGAACCTCAT?CACGGGCATC?GAGGTGATCG

TCTCTAGAGG?AAACTGGGGC?TCTTGGAGTA?GTGCCCGTAG?CTCCACTAGC

·?E??G??W???W??R??G???Y??G??P??D???G??H??F???G??M1551???ACGAAGGCTG?GTGGCGTGGC?TATGGGCCGG?ATGGCCATTT?TGGCATGTTC

TGCTTCCGAC?CACCGCACCG?ATACCCGGCC?TACCGGTAAA?ACCGTACAAG

P??A??N??Y???V??E??L???I??E??*???G??*??G??H???I??L1601???CCTGCCAACT?ACGTGGAGCT?CATTGAGTGA?GGCTGAGGGC?ACATCTTGCC

GGACGGTTGA?TGCACCTCGA?GTAACTCACT?CCGACTCCCG?TGTAGAACGG

·F??P??S???Q??T??W??L???P??Y??C???W??K??R???R??P1651???CTTCCCCTCT?CAGACATGGC?TTCCTTATTG?CTGGAAGAGG?AGGCCTGGGA

GAAGGGGAGA?GTCTGTACCG?AAGGAATAAC?GACCTTCTCC?TCCGGACCCT

·?*??H??S???A??L??F???Q??E??*??D???P??Q??*???G??*1701???GTTGACATTC?AGCACTCTTC?CAGGAATAGG?ACCCCCAGTG?AGGATGAGGC

CAACTGTAAG?TCGTGAGAAG?GTCCTTATCC?TGGGGGTCAC?TCCTACTCCG

L??R??A??P???S??G??L???A??D??S???A??C??H??P???K??C1751???CTCAGGGCTC?CCTCCGGCTT?GGCAGACTCA?GCCTGTCACC?CCAAATGCAG

GAGTCCCGAG?GGAGGCCGAA?CCGTCTGAGT?CGGACAGTGG?GGTTTACGTC

·N??G??L???V??I??P??T???H??P??S???C??I??P???R??P1801???CAATGGCCTG?GTGATTCCCA?CACATCCTTC?CTGCATCCCC?CGACCCTCCC

GTTACCGGAC?CACTAAGGGT?GTGTAGGAAG?GACGTAGGGG?GCTGGGAGGG

T??A??W???L??L??P???L??T??G??Y???*??A??K???P??C1851???AGACAGCTTG?GCTCTTGCCC?CTGACAGGAT?ACTGAGCCAA?GCCCTGCCTG

TCTGTCGAAC?CGAGAACGGG?GACTGTCCTA?TGACTCGGTT?CGGGACGGAC

W??P??S??P???E??W??P???L??P??S???C??G??E??G???S??*1901???TGGCCAAGCC?CTGAGTGGCC?ACTGCCAAGC?TGCGGGGAAG?GGTCCTGAGC

ACCGGTTCGG?GACTCACCGG?TGACGGTTCG?ACGCCCCTTC?CCAGGACTCG

·G??A??S???G??R??L??W???L??P??S???A??F??I???C??L1951???AGGGGCATCT?GGGAGGCTCT?GGCTGCCTTC?TGCATTTATT?TGCCTTTTTT

TCCCCGTAGA?CCCTCCGAGA?CCGACGGAAG?ACGTAAATAA?ACGGAAAAAA

·??F??S??L??A??S??K???G??W??W??P???P??L??F???R??M2001???CTTTTTCTCT?TGCTTCTAAG?GGGTGGTGGC?CACCACTGTT?TAGAATGACC

GAAAAAGAGA?ACGAAGATTC?CCCACCACCG?GTGGTGACAA?ATCTTACTGG

L??G??N??S???E??R??R???E??L??F???L??A??E??F???V??T2051???CTTGGGAACA?GTGAACGTAG?AGAATTGTTT?TTAGCAGAGT?TTGTGACCAA

GAACCCTTGT?CACTTGCATC?TCTTAACAAA?AATCGTCTCA?AACACTGGTT

·V??R??V???D??H??G??G???L??A??A???G??N??L???S??C2101???AGTCAGAGTG?GATCATGGTG?GTTTGGCAGC?AGGGAATTTG?TCTTGTTGGA

TCAGTCTCAC?CTAGTACCAC?CAAACCGTCG?TCCCTTAAAC?AGAACAACCT

·?L??L??C???A??P??H???S??I??S??L???S??L??C???L??G2151???GCCTGCTCTG?TGCTCCCCAC?TCCATTTCTC?TGTCCCTCTG?CCTGGGCTAT

CGGACGAGAC?ACGAGGGGTG?AGGTAAAGAG?ACAGGGAGAC?GGACCCGATA

G??K??W??G???C??R??W???P??S??S???H??P??G??Y???S??K2201???GGGAAGTGGG?GATGCAGATG?GCCAAGCTCC?CACCCTGGGT?ATTCAAAAAC

CCCTTCACCC?CTACGTCTAC?CGGTTCGAGG?GTGGGACCCA?TAAGTTTTTG

·A??D??T???T??C??S??S???T??R??L???T??R??C???L??Q2251???GGCAGACACA?ACATGTTCCT?CCACGCGGCT?CACTCGATGC?CTGCAGGCCC

CCGTCTGTGT?TGTACAAGGA?GGTGCGCCGA?GTGAGCTACG?GACGTCCGGG

·?V??C??A???S??T??D???S??D??F??R???K??S??K???K??K2301???CAGTGTGTGC?CTCAACTGAT?TCTGACTTCA?GGAAAAGTAA?AAAAAAAAAA

GTCACACACG?GAGTTGACTA?AGACTGAAGT?CCTTTTCATT?TTTTTTTTTT

K??K??L??E???K??L??W???T??S??S2351???AAAAAACTCG?AGAAGCTTTG?GACTTCTTCG?CCA

TTTTTTGAGC?TCTTCGAAAC?CTGAAGAAGC?GGT

Table 4

The MannC aminoacid sequence of inferring

I??R??H???E??H??G????E??E?T??*???E??V??*???N??K1??????GAATTCGGCA?CGAGCACGGT?GAAGAGACAT?GAGAGGTGTA?GAATAAGTGG

CTTAAGCCGT?GCTCGTGCCA?CTTCTCTGTA?CTCTCCACAT?CTTATTCACC

E??A??P??G???A??P??P???V??S??P???R??G??A??R???G??G51?????GAGGCCCCCG?GCGCCCCCCC?GGTGTCCCCG?CGAGGGGCCC?GGGGCGGGGT

CTCCGGGGGC?CGCGGGGGGG?CCACAGGGGC?GCTCCCCGGG?CCCCGCCCCA

·R??R??P???C??G??P??P???V??K??Y???H??Y??S???D??R101????CCGCCGGCCC?TGCGGGCCGC?CGGTGAAATA?CCACTACTCT?GATCGTTTTT

GGCGGCCGGG?ACGCCCGGCG?GCCACTTTAT?GGTGATGAGA?CTAGCAAAAA

·?T??D??P???V??R??R???G??G??E??P???R??G??A???L??A151????TCACTGACCC?GGTGAGGCGG?GGGGGCGAGC?CCCGAGGGGC?TCTCGCTTCT

AGTGACTGGG?CCACTCCGCC?CCCCCGCTCG?GGGCTCCCCG?AGAGCGAAGA

G??A??K??R???P??A??A???R??R??P???G??A??T??R???S??G201????GGCGCCAAGC?GCCCGGCCGC?GCGCCGGCCG?GGCGCGACCC?GCTCCGGGGA

CCGCGGTTCG?CGGGCCGGCG?CGCGGCCGGC?CCGCGCTGGG?CGAGGCCCCT

·S??A??R???W??G??V??*???L??G??R???Y??T??C???Q??T251????CAGTGCCAGG?TGGGGAGTTT?GACTGGGGCG?GTACACCTGT?CAAACGGTAA

GTCACGGTCC?ACCCCTCAAA?CTGACCCCGC?CATGTGGACA?GTTTGCCATT

·?Q??V??S???*??G??E???L??R??E??D???R??N??L???P??W301????CGCAGGTGTC?CTAAGGCGAG?CTCAGGGAGG?ACAGAAACCT?CCCGTGGAGC

GCGTCCACAG?GATTCCGCTC?GAGTCCCTCC?TGTCTTTGGA?GGGCACCTCG

R??R??A??K???A??R??L???I??L??I???F??S??T??N???T??D351????AGAAGGGCAA?AAGCTCGCTT?GATCTTGATT?TTCAGTACGA?ATACAGACCG

TCTTCCCGTT?TTCGAGCGAA?CTAGAACTAA?AAGTCATGCT?TATGTCTGGC

·E??S??G???A??S??R??S???F??*??P???F??G??F???*??A401????TGAAAGCGGG?GCCTCACGAT?CCTTCTGACC?TTTTGGGTTT?TAAGCAGGAG

ACTTTCGCCC?CGGAGTGCTA?GGAAGACTGG?AAAACCCAAA?ATTCGTCCTC

·?V??R??K???V??T??T???G??I??T??G???L??W??R???P??S451????GTGTCAGAAA?AGTTACCACA?GGGATAACTG?GCTTGTGGCG?GCCAAGCGTT

CACAGTCTTT?TCAATGGTGT?CCCTATTGAC?CGAACACCGC?CGGTTCGCAA

H??S??D??V???A??F??*???S??F??D???V??G??S??S???Y??H501????CATAGCGACG?TCGCTTTTTG?ATCCTTCGAT?GTCGGCTCTT?CCTATCATTG

GTATCGCTGC?AGCGAAAAAC?TAGGAAGCTA?CAGCCGAGAA?GGATAGTAAC

·E??A??E???F??T??K??R???W??I??V???H??P??L???I??G551????TGAAGCAGAA?TTCACCAAGC?GTTGGATTGT?TCACCCACTA?ATAGGGAACG

ACTTCGTCTT?AAGTGGTTCG?CAACCTAACA?AGTGGGTGAT?TATCCCTTGC

·?S??W??V???*??T??V???V??R??Q??V???S??F??T???L??L601????TGAGCTGGGT?TTAGACCGTC?GTGAGACAGG?TTAGTTTTAC?CCTACTGATG

ACTCGACCCA?AATCTGGCAG?CACTCTGTCC?AATCAAAATG?GGATGACTAC

M??C??C??C???H??G??N???P??A??Q???Y??E??R??N???R??R651????ATGTGTTGTT?GCCATGGTAA?TCCTGCTCAG?TACGAGAGGA?ACCGCAGGTT

TACACAACAA?CGGTACCATT?AGGACGAGTC?ATGCTCTCCT?TGGCGTCCAA

·R??H??L???V??Y??V??L???G??*??G???A??N??G???A??K701????CAGACATTTG?GTGTATGTGC?TTGGCTGAGG?AGCCAATGGG?GCGAAGCTAC

GTCTGTAAAC?CACATACACG?AACCGACTCC?TCGGTTACCC?CGCTTCGATG

·?S??V??G???L??*??L???N??A??S??K???S??E??S???R??P751????CATCTGTGGG?ATTATGACTG?AACGCCTCTA?AGTCAGAATC?CCGCCCAGGC

GTAGACACCC?TAATACTGAC?TTGCGGAGAT?TCAGTCTTAG?GGCGGGTCCG

G??T??I??R???Q??R??R???G??A??S???V??G??L??G???*??P801????GGAACGATAC?GGCAGCGCCG?CGGAGCCTCG?GTTGGCCTCG?GATAGCCGGT

CCTTGCTATG?CCGTCGCGGC?GCCTCGGAGC?CAACCGGAGC?CTATCGGCCA

·P??R??L???S??P??P??A???G??R??P???P??P??S???T??R851????CCCCCGCCTG?TCCCCGCCGG?CGGGCCGCCC?CCCCCCCTCC?ACGCGCCCCG

GGGGGCGGAC?AGGGGCGGCC?GCCCGGCGGG?GGGGGGGAGG?TGCGCGGGGC

·?R??A??G???G??R??V???P??R??R??A???P??G??P???G??S901????CGCGCGCGGG?AGGGCGCGTG?CCCCGCCGCG?CGCCGGGACC?GGGGTCCGGT

GCGCGCGCCC?TCCCGCGCAC?GGGGCGGCGC?GCGGCCCTGG?CCCCAGGCCA

A??E??C??P???S??S??W???E??T??G???R??G??R??K???G??G951????GCGGAGTGCC?CTTCGTCCTG?GGAAACGGGG?CGCGGCCGGA?AAGGCGGCCG

CGCCTCACGG?GAAGCAGGAC?CCTTTGCCCC?GCGCCGGCCT?TTCCGCCGGC

·P??L??A???R??H??A??P???H??V??R???A??R??A???E??F1001???CCCCCTCGCC?CGTCACGCAC?CGCACGTTCG?TGCTCGTGCC?GAATTCGGCA

GGGGGAGCGG?GCAGTGCGTG?GCGTGCAAGC?ACGAGCACGG?CTTAAGCCGT

·?S??S??T???I??H??N???R??H??T??S???A??C??I???F??M1051???CGAGTAGCAC?CATTCACAAT?AGACATACAA?GTGCATGTAT?CTTTATGATA

GCTCATCGTG?GTAAGTGTTA?TCTGTATGTT?CACGTACATA?GAAATACTAT

*??*??I??L???F??L??W???V??D??I???Q??*??W??D???C??*1101???TAATGAATTC?TTTTCCTTTG?GGTAGATATC?CAGTAGTGGG?ATTGCTAGAT

ATTACTTAAG?AAAAGGAAAC?CCATCTATAG?GTCATCACCC?TAACGATCTA

·T??W??*???F??Y??F??W???F??I??E???K??S??S???Y??*1151???CACCTGGTAG?TTCTATTTCT?GGTTTATTGA?GAAATCTTCA?TACTGATTTC

GTGGACCATC?AAGATAAAGA?CCAAATAACT?CTTTAGAAGT?ATGACTAAAG

·?*??R??L???Y??K??F???T??S??L??P???S??D??F???F??K1201???CATAGAGGTT?GTACAAATTT?ACATCCCTAC?CAAGTGATTT?TTTTAAATAT

GTATCTCCAA?CATGTTTAAA?TGTAGGGATG?GTTCACTAAA?AAAATTTATA

E??R??M??V???W??R??N???A??P??H???*??Y??P??P???F??T1251???GAAAGAATGG?TCTGGAGAAA?TGCCCCTCAT?TAGTATCCCC?CTTTTACCTC

CTTTCTTACC?AGACCTCTTT?ACGGGGAGTA?ATCATAGGGG?GAAAATGGAG

·L??L??Q???N??D??F??K???G??Y??R???Y??L??Q???V??S1301???TCTACTGCAG?AATGACTTCA?AGGGGTACAG?GTATTTACAA?GTTTCATTAT

AGATGACGTC?TTACTGAAGT?TCCCCATGTC?CATAAATGTT?CAAAGTAATA

·?R??Q??I???E??Y??*???N??F??C??I???R??G??T???D??F1351???ACAGACAAAT?TGAATATTGA?AATTTCTGCA?TAAGAGGCAC?AGATTTTAGG

TGTCTGTTTA?ACTTATAACT?TTAAAGACGT?ATTCTCCGTG?TCTAAAATCC

I??Q??S??C???M??N??K???D??K??C???S??R??D??L???Q??S1401???ATTCAAAGTT?GTATGAACAA?GGACAAGTGC?TCTAGGGACT?TGCAAAGCTG

TAAGTTTCAA?CATACTTGTT?CCTGTTCACG?AGATCCCTGA?ACGTTTCGAC

·N??W??K???S??Q??M??K???Y??I??S???S??S??T???T??S1451???GAATTGGAAA?TCTCAGATGA?AATACATTTC?TAGTAGTACC?ACCAGCATAT

CTTAACCTTT?AGAGTCTACT?TTATGTAAAG?ATCATCATGG?TGGTCGTATA

·?S??T??E???L??A??L???*??S??S??L???I??P??T???Y??*1501???ATTCTACTGA?ATTGGCTTTG?TGATCATCAT?TAATACCTAC?TTATTAAAAC

TAAGATGACT?TAACCGAAAC?ACTAGTAGTA?ATTATGGATG?AATAATTTTG

*??*??K??G???F??I??S???N??I??L???*??G??I??K???I??K1551???TAATGAAAAG?GGTTTATATC?AAATATACTT?TAAGGTATAA?AAATCAAATT

ATTACTTTTC?CCAAATATAG?TTTATATGAA?ATTCCATATT?TTTAGTTTAA

·*??V??K???L??F??S??L???A??F??*???F??Q??N???I??K1601???ATAGGTAAAG?CTGTTTTCTT?TAGCATTTTA?ATTTCAAAAC?ATAAAATAGC

TATCCATTTC?GACAAAAGAA?ATCGTAAAAT?TAAAGTTTTG?TATTTTATCG

·?P??S??I???G??H??L???Y??C??T??R???H??C??V???C??H1651???TACCGTCTAT?TGGGCATTTA?TACTGTACCA?GACACTGTGT?TTGTCACATT

ATGGCAGATA?ACCCGTAAAT?ATGACATGGT?CTGTGACACA?AACAGTGTAA

S??K??M??F???S??W??*???C??S??Q???*??F??C??R???V??R1701???TCAAAAATGT?TCTCATGGTA?ATGTTCACAA?TAATTCTGTA?GGGTGAGAAA

AGTTTTTACA?AGAGTACCAT?TACAAGTGTT?ATTAAGACAT?CCCACTCTTT

·S??L??T???V??V??R??L???F??S??K???R??N??L???*??T1751???TAGTCTTACC?GTAGTAAGAC?TATTCAGTAA?ACGAAACCTC?TGAACCTTGG

ATCAGAATGG?CATCATTCTG?ATAAGTCATT?TGCTTTGGAG?ACTTGGAACC

·?F??N??L???R??K??V???S??N??R??T???R??T??*???T??*1801???AGTTCAACTT?GCGCAAAGTT?AGTAACAGGA?CTAGGACTTG?AACCTGAACC

TCAAGTTGAA?CGCGTTTCAA?TCATTGTCCT?GATCCTGAAC?TTGGACTTGG

I??T??L??Q???I??S??P???Y??H??T???A??S??T??C???A??C1851???ATCACACTCC?AGATCTCTCC?ATACCACACT?GCTAGCACAT?GTGCCTGTCA

TAGTGTGAGG?TCTAGAGAGG?TATGGTGTGA?CGATCGTGTA?CACGGACAGT

·L??I??P???G??S??C??Y???F??P??F???Y??F??L???S??L1901???TCTTATTCCT?GGCTCCTGTT?ATTTCCCTTT?TTATTTCCTT?TCCCTTCCTC

AGAATAAGGA?CCGAGGACAA?TAAAGGGAAA?AATAAAGGAA?AGGGAAGGAG

·?T??T??P???F??S??P???H??F??F??S???F??F??L???I??V1951???CCACAACCCC?TTTTTCCCCC?CATTTCTTTT?CTTTCTTTTT?AATTGTTAAT

GGTGTTGGGG?AAAAAGGGGG?GTAAAGAAAA?GAAAGAAAAA?TTAACAATTA

Y??I??T??N???T??C??L???S??E??Q???L??I??*??H???K??R2001???TACATAACTA?ATACATGCTT?ATCAGAACAA?TTGATATAGC?ACAAAAGGAT

ATGTATTGAT?TATGTACGAA?TAGTCTTGTT?AACTATATCG?TGTTTTCCTA

·*??S??T???G??E??*??*???L??I??P???V??I??L???A??L2051???ATAAAGTACG?GGTGAGTGAT?AGCTCATCCC?TGTAATCCTA?GCACTTTGGA

TATTTCATGC?CCACTCACTA?TCGAGTAGGG?ACATTAGGAT?CGTGAAACCT

·?A??K??A???G??R??S???L??E??S??R???V??R??D???Q??P2101???AGGCCAAGGC?AGGCAGATCA?CTTGAGTCCA?GAGTTCGAGA?CCAGCCTGGG

TCCGGTTCCG?TCCGTCTAGT?GAACTCAGGT?CTCAAGCTCT?GGTCGGACCC

Q??H??G??E???T??L??S???L??Q??K???N??T??K??I???*??P2151???CAACATGGTG?AAACCCTGTC?TCTACAAAAA?AATACAAAAA?TTTAGCCGGG

GTTGTACCAC?TTTGGGACAG?AGATGTTTTT?TTATGTTTTT?AAATCGGCCC

·V??L??A???H??T??C??S???L??S??Y???S??E??G???*??G2201???CGTGCTGGCA?CACACCTGTA?GTCTCAGCTA?CTCTGAGGGC?TGAGGTGGGA

GCACGACCGT?GTGTGGACAT?CAGAGTCGAT?GAGACTCCCG?ACTCCACCCT

·?I??D??*???A??Q??E???V??E??A??A???A??V??R???*??D2251???AGATTGATTG?AGCCCAGGAG?GTGGAAGCTG?CAGCAGTGCG?CTGAGATTGC

TCTAACTAAC?TCGGGTCCTC?CACCTTCGAC?GTCGTCACGC?GACTCTAACG

A??I??A??L???Q??P??G???*??E??R???E??T??L??S???Q??K2301???GCCATTGCAC?TCCAGCCTGG?GTGAGAGAGA?GAGACCCTGT?CTCAAAAAAA

CGGTAACGTG?AGGTCGGACC?CACTCTCTCT?CTCTGGGACA?GAGTTTTTTT

·K2351???AAAAA

TTTTT

Table 5

Relatively

MannA.MammB.MannC. prostate gland

Claims (7)

1. breast statin sample epithelial cell growth inhibitors, at least three of this molecule/one suppresses to have significant sequence homogeny with breast.

2. inhibitor according to claim 1, wherein epithelial cell is central nervous system, heart, small intestine, large intestine, appendix, rectum, lymphocyte, medullary cell, lung and air flue, bladder, uterus, prostate gland, testis, ovary, liver, pancreas, suprarenal gland, sialisterium and mammary gland.

3. the nucleotide sequence of the epithelial cell growth inhibitors of encoding, this inhibitor suppresses the epithelial growth of non-breast, and wherein this nucleotide sequence can obtain from normal non-breast epithelial cell amplification with the breast statin inside PCR primer that obtains of deriving.

4. nucleotide sequence according to claim 3, wherein epithelial cell is colon, ovary, prostate gland, spleen, testis or thymocyte.

5. method that detects cell carcinoma, this method comprises:

Whether there are the described protein of claim 1 or the described nucleotide sequence of claim 3 or its content in the analysing body fluid; With

The minimizing or the disappearance of described protein or described nucleotide sequence are associated with cell carcinoma.

6. method for the treatment of patient's cell carcinoma, this method comprises:

Give described protein of patient's claim 1 or the described nucleic acid of claim 3.

7. diagnostic kit, it comprises:

Described protein of claim 1 and/or the described nucleotide sequence of claim 4; And optional specificity is in conjunction with the described proteinic antibody of claim 1.

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