CN1957088A - Method of judging properties of mammalian cell and method of diagnosing cancer - Google Patents
Method of judging properties of mammalian cell and method of diagnosing cancer Download PDFInfo
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Abstract
CDK profile such as the specific activity ratio of two cyclin-dependent kinases closely correlates to the grade of malignancy and the presence/absence of the sensitivity to a stimulating substance such as an anticancer agent of a cancer tissue in clinical medicine. Therefore, the properties of a mammalian cell such as the grade of malignancy of cancer, cell growth ability and the sensitivity to a stimulating substance such as an anticancer agent can be judged based on its CDK profile or by comparing the specific activity ratio of two CDKs with a definite threshold. This sensitivity judgment method shows a high ratio of correct answers particularly in positive sensitivity and is useful in judging the efficacy of an anticancer drug therapy.
Description
Technical field:
The present invention relates to a kind of authentication method and with the method for this authentication method diagnosing cancer.This authentication method is identified the grade of malignancy of cancer and tumour according to the ratio of the activity value of the specific activity of cyclin-dependent kinase (CDK) etc., CDK and expression amount and to the susceptibility of excitor substances such as anticarcinogen.
Background technology:
The method of diagnosing cancer has serum diagnostics, organizational diagnosis's method of making biopsy and the cytologic diagnosis method of checking the tumor marker in the serum clinically now.
So-called tumor marker refers to that the individual gene that take place, do not show show and the materials such as albumen, glycoprotein and lipid that form in tumour cell in ripening process, serum diagnostics is to judge the stadium of tumour and grade of malignancy by finding tumor marker.Yet the most cancer specific of tumor marker that uses is not too high now, and early cancer is expressed very little, and the diagnosis susceptibility is low.Therefore, present situation is not too high at medical scene by the reliability of tumor marker diagnosing cancer.
On the other hand, to be a kind of pathologist and cell screening person (cellsScreener) will use microscopic examination behind the biological tissue section of sampling and the cell dyeing for organizational diagnosis's method and cytologic diagnosis method, the method for identifying with the experience of oneself according to its form and painted appearance.But because staining is different because of medical institutions, identify that finally person individual's experience is made according to the observation, the delicate stage at middle differentiated and initial stage is difficult to make a definite diagnosis, and falsely drops that example that therapeutics causes developing into the severe cancer also more a generation.
In view of this, designed the method that TNM classifies and unifies to identify, rather popular in the world.The TNM classification of cancer is the performance malignant tumour progress degree methods that International Union of Counter Cancer (UICC) adopts, and " T " refers to the size of primary tumo(u)r, and " N " expression regional nodes shifts, and " M " represents distant metastasis.Wherein about " T ", degree of progress is divided into 1 (tumour is confined to in-house degree)～4 (tumour has the performance at organizational shape one-tenth), about " N ", its progress degree is divided into 0 (not finding metastases in local lymph node)～3 (confirming nodus lymphoideus transferring rate on histology), and " M " then degree of progress is divided into 0 (no distant metastasis)～1 (distant metastasis is arranged).At T, N, M whichsoever all is that numeral is big more, and prognosis is poor more, and grade of malignancy is high more.
This TNM classification is all useful to decision treatment plan and judging prognosis, therefore is widely used.But it also has very big defective clinically, at present, is judged as the patient of early cancer by the TNM classification, and the words of mammary cancer have in the patient 5 years of 10-20% approximately owing to distant metastasis causes recurrence, death.And, although can hold the state of an illness when diagnosing, still can't correctly predict prognosis based on the judgement of TNM classification.
Except that above-mentioned authentication method, because of in cancerous tissue, having triploid, tetraploid equimultiple somatocyte in a large number, be conceived to this, also useful fluorescent mark formula separator cell (FACS) is measured dna content, is diagnosed the method for cancer grade of malignancy from this measurement result.Yet, the tissue of gathering is placed on facs analysis and must be processed into one by one unicellular earlier, and being processed into unicellular group, the biopsy sample generally is not easy to, need masterful technique.Therefore, also relatively more difficult now with FACS diagnosis cancer in the on-the-spot practicability of medical treatment.
Because above-mentioned reality, people wish to have the such authentication method that can make a definite diagnosis: it does not require the specimen preparation of this trouble of unicellularization, and reduce clinically because of everyone judgement and the different diagnostic results that cause of the determination methods of medical institutions different.
In recent years, using instrument to carry out molecular diagnosis receives publicity as rare unified authentication method because of the different cancer of diagnostician's difference result.
The useful DNA section of molecular diagnosis is compared with regard to the expression and the standard of its gene transcript, method according to comparative result diagnosis cancer grade of malignancy, but, because only the dependency of the protein expression that shows in the expression of a transcript and the biology is not high, therefore often hold the suspicious attitude to its validity in clinical scene.
On the other hand, the R﹠D work of carrying out molecular diagnosis according to the protein that shows in the biology is carried out.Having proposed with the CDK1 of sample and CDK4 expression amount, necessary words such as, patent documentation 1 is the method that index is diagnosed with the halmatogenesis state of P53 also.Patent documentation 2 proposes to be expressed as the method that index is carried out cancer and preceding cancer cells condition diagnosing with the surplus of CDK4, CDK6 and cyclin-dependent kinase inhibitor (CDK inhibitor).
Though being expressed in of well-known cyclin-dependent kinase (CDK) induced and very high in the proliferating cells by multiplicaiton factor, but generally in cell, all exist a certain amount of, they are by activation such as phosphorylations, combine the mixture that generates CDK and cycle element with plain molecule of cycle, so all demonstrate activity in the specified phase of corresponding cell cycle of various CDK.And people know that also the CDK inhibitor can be attached to CDK and/or plain CDK mixture of cycle hinders the CDK activity.So, the practical adjustments cell cycle is very complicated, therefore, in the diagnosis based on the expression amount of CDK, cycle element and CDK inhibitor only, is not enough as the index of regulation of Cell Cycle state.
As the curative effect index of anticancer drug therapy, wish to know the anti-cancer drugs susceptibility of tumour cell sometimes from vitro culture.The testing method of external anti-tumor medicine susceptibility is such as mtt assay that has people to be familiar with and DISC method.So-called mtt assay utilizes exactly that (3-(4 as MTT, 5-dimcthylthioazol-2-yl)-2,5-diphenyl-tetrazolium bromide, tetramethyl-azo azoles salt) when putting into viable cell, reductase enzyme in the viable cell plastosome (succinodehydrogenase) can make MTT salt split dead cell then can not principle, on the basis of adding MTT and tested medicine, cultivated cancer cells 3 hours, measure its absorbancy (succinic dehydrogenase activity), quantitatively viable count.It was reported by (non-patent literature 1): use this method, when in fact contrast has or not recurrence with anticancer drug therapy, accuracy (accuracy) is 80%, wherein in vitro tests is diagnosed as anti-cancer drugs susceptibility (positive prediction), and the accuracy that reality does not have recurrence (positive prediction rate) by anticancer drug therapy is 68%.
Differential staining (DISC method) is that cancer cells was immersed in the chemotherapeutics four days, by fast green dead cell is painted, hematoxylin eosin viable cell is dyeed, and differentiates the life and death of cancer cells.It was reported by (non-patent literature 1) that its accuracy (accuracy) is 82.7%, the accuracy of positive prediction rate is 62.5%.
Above-mentioned which kind of method no matter, its positive prediction rate is less than 70%, as judging whether that it is not enough taking the index of cancer drug therapy.
Patent documentation 1: specially show 2002-504683 number
Patent documentation 2: specially show 2002-519681 number
Patent documentation 3: specially open 2002-335997 number
Non-patent literature 1:Weisenthal, the present situation of L.M.and Nygren.P (2002) cell cultures resistant test (Current status of cell culture drug resistance testing (CCDRT)),
Http:// weisenthal.org/oncol t.htm
Summary of the invention:
The problem that invention will solve:
The present invention In view of the foregoing researches and develops, its purpose is, the grade of malignancy of clinical judgment cancer cells (easily metastatic rate, easily degree of recurrence, prognosis mala degree), a kind of authentication method that can correctly stably make evaluation is provided and cancer is carried out the method for molecular diagnosis, to eliminate because of medical institutions and the different phenomenon of the individual different diagnostic results that bring with this method.
The present invention is also for providing the drug sensitive test in the past of a kind of ratio more accurate determination methods with regard to mammalian cell to the judgement of excitor substance susceptibility such as anticarcinogen, makes it can be as the judge index of whether carrying out chemotherapy such as anticancer drug therapy.
The means that the present invention deals with problems
Inventors of the present invention are conceived to the specific activity of cyclin-dependent kinase, the relation that itself and histopathology are judged various discussion have been carried out, found that, comprising the multiplication capacity of the CDK profile of the specific activity of two kinds of cyclin-dependent kinases (CDK) or its inverse number and cell and organizing between the grade of malignancy (easily degree of recurrence) has high correlation, has finished character authentication method of the present invention.In addition, about the susceptibility of Mammals to excitor substance, also finding has dependency with two kinds of CDK profiles, has finished susceptibility authentication method of the present invention.
The authentication method of mammalian cell character of the present invention is the activity value and the activity value of element dependant kinase second cycle when (the 2nd CDK) of expression amount and the CDK profile of the ratio of expression amount that contains plain dependant kinase of period 1 (CDK) according to mammalian cell, diagnoses above-mentioned mammalian cell character.
The CDK profile of above-mentioned CDK profile and standard cell lines is made comparisons, also can make above-mentioned character and identify.
As a kind of mode based on the authentication method of above-mentioned CDK profile, also the ratio of the ratio (A2) of the activity value of the ratio (A1) of the activity value of plain dependant kinase of period 1 of mammalian cell (CDK) and expression amount and plain dependant kinase second cycle (the 2nd CDK) and expression amount and certain threshold value corresponding to this ratio can be compared, above-mentioned mammalian cell character is identified.
Above-mentioned cyclin-dependent kinase preferably is selected from CDK1, CDK2, CDK4, CDK6, plain A dependant kinase of cycle, plain B dependant kinase of cycle and plain D dependant kinase of cycle, the preferred above-mentioned period 1, plain dependant kinase was CDK1, and second cycle, plain dependant kinase was CDK2.
Above-mentioned character both can be the multiplication capacity of above-mentioned mammalian cell, also can be the grade of malignancy of mammalian cell.
Above-mentioned grade of malignancy comprises: easily metastatic rate, easily degree of recurrence, prognosis mala degree.Above-mentioned transfer comprises nodus lymphoideus transferring rate and distant metastasis.
Method for cancer diagnostics of the present invention is with above-mentioned authentication method diagnosing cancer grade of malignancy of the present invention to the cell gathered from biological tissue.
Mammalian cell of the present invention then is the activity value and the activity value of element dependant kinase second cycle when (the 2nd CDK) of expression amount and the CDK profile of the ratio of expression amount that contains plain dependant kinase of period 1 (CDK) according to mammalian cell to the susceptibility authentication method of excitor substance, identifies the susceptibility of above-mentioned cell to excitor substance.
A kind of embodiment as susceptibility authentication method of the present invention, be that the ratio of the ratio (A2) of the activity value of the ratio (A1) of the activity value of plain dependant kinase of period 1 (CDK) and expression amount and plain dependant kinase second cycle (the 2nd CDK) and expression amount and certain threshold value corresponding to this ratio are compared, identify the susceptibility of above-mentioned mammalian cell excitor substance.
Preferably the ratio (A2) of the activity value of the ratio (A1) of the activity value of plain dependant kinase of period 1 (CDK) and expression amount and/or plain dependant kinase second cycle (the 2nd CDK) and expression amount is self-correspondingly decided threshold value and compared respectively with each, identify the susceptibility of above-mentioned mammalian cell excitor substance.
Above-mentioned cyclin-dependent kinase preferably is selected from CDK1, CDK2, CDK4, CDK6, plain A dependant kinase of cycle, plain B dependant kinase of cycle and plain D dependant kinase of cycle.
Above-mentioned excitor substance preferably is selected from multiplicaiton factor, anticarcinogen and variation originality material.
In addition, the said CDK profile of this specification sheets refers to comprise the information of following content: the ratio (such as specific activity) of the activity value of at least a CDK that certain cell has and expression amount and/or the numerical value that calculates from a plurality of CDK activity values and expression amount (such as ratio (as A1/A2 or A2/A1) of: the ratio (A1) of the activity value of a CDK and expression amount and the ratio (A2) of the activity value of the 2nd CDK and expression amount etc.).
The said cyclin-dependent kinase of this specification sheets (CDK) specific activity refers to the enzymic activity of a certain amount of every kind of enzyme, the value of trying to achieve by following formula.
CDK specific activity=CDK activity value/CDK expression amount
So-called CDK activity value is based on by the unit of the amount of substrate of phosphorylation in the formula, is the numerical value (relative reactivity value) of trying to achieve with the activity value of the measured value of actual celliferous sample and standard substance.So-called CDK expression amount refers to be present in the amount (molecule number unit) of the CDK in the actual celliferous sample.
Easy metastatic rate of the present invention refers to that cancer cells transferred at a distance tissue, set up the probability height that shifts nest by former focus.The biological property of the cancer cells that known probability is high (easily metastatic rate) can, move and can represent with soaking into.
So-called easily recurrence degree refers to, the cancer patients group's who classifies according to certain classification (such as the phase classification) recurrence frequency.Such as: III phase recurrence rate 50%, than easily recurrence of II phase (recurrence rate 20%).
The prognosis mala degree refers to according to the cancer patients group of certain classification (such as phase classification) classification 5 years or 10 years with interior dead frequency.Such as: III phase mortality ratio 50%, easily more dead than the II phase (mortality ratio 20%), prognosis mala.
The invention effect:
Mammalian cell character authentication method of the present invention can be by being that index avoids the sum of errors that brings because of the compound method of measuring sample uneven with the specific activity.That the present invention uses as identification of indicator, with reference to the ratio of CDK specific activity profile, particularly two kinds of CDK specific activitys of two kinds of CDK specific activitys, exist the property correlation of cancers such as ratio and dysploidy very high with the cell that is present in the cell cycle, therefore can carry out the very high Molecular Identification of confidence level.In addition, owing to used index based on the cell cycle, can not only identify the character and the proterties of present cytobiology, can also identify to clinical character (representative is grade of malignancy) and to the susceptibility even the patience of medicaments such as anticarcinogen and outside stimulus.Particularly about the evaluation of susceptibility, it is very high to be accredited as its accuracy rate of susceptibility (positive), and it is very outstanding pretending the judge index of implementing anticancer drug therapy for decision.
Description of drawings:
Fig. 1 is the explanatory view of cell cycle.
Fig. 2 is the demonstration figure of the measurement result of various culturing cell dna contents.
Fig. 3 is the explanatory view of dna content data results.
Fig. 4 is the demonstration figure that there be ratio of various culturing cells in the S phase.
Fig. 5 is that the CDK1 specific activity of various culturing cells and the measurement result of CDK2 specific activity show figure.
Fig. 6 picks up from the CDK specific activity profile demonstration figure that is diagnosed as a low-risk corpse or other object for laboratory examination and chemical testing in patient with breast cancer's the tissue.
Fig. 7 picks up from the CDK specific activity profile demonstration figure that is diagnosed as a high risk corpse or other object for laboratory examination and chemical testing in patient with breast cancer's the tissue.
Fig. 8 is that the CDK1 specific activity of patients with mastocarcinoma A-I and the measurement result of CDK2 specific activity show figure.Patients with mastocarcinoma A-I accepts the treatment of anticarcinogen polyenoid taxol, and has accepted the investigation of dispensing back tumour size variation.
Fig. 9 is that the CDK1 specific activity of patients with mastocarcinoma J-P and the measurement result of CDK2 specific activity show figure.Patients with mastocarcinoma J-P accepts the treatment of anticarcinogen taxol, and has accepted the investigation of dispensing back tumour size variation.
The specific embodiment of the invention:
The method of evaluation mammalian cell character of the present invention is to measure the expression amount and the activity value of cyclin-dependent kinase more than two kinds of mammalian cell, according to the activity value that contains plain dependant kinase of period 1 and expression amount second cycle when plain dependant kinase the CDK profile of activity value and the ratio of expression amount, identify the character of this mammalian cell.Authentication method of the present invention is applied in the tissue that contains tumour cell can the diagnosing tumour cell character and the grade of malignancy of cancer.
Authentication method of the present invention there is no specific as the Mammals of object, but its to the people particularly clinical state need more specifically concerning cancer is made the people of diagnosis of great use.
The inventive method as the cell of object so long as to constitute mammiferous biological tissue be sustentacular tissues such as fibering conjunctive tissue, cartilaginous tissue, osseous tissue, blood and lymph; Epithelium; The cell of muscle tissue and nervous tissue gets final product, but for individual being in harmonious proportion, giving tumour cell that the propagation controlling organization brings unusual tissue etc. to expect that the cell of pathological information is particularly suitable such as breaking.Such as: the tumour cell that is grown in positions such as mammary gland, lung, liver, stomach, large intestine, pancreas, skin, uterus, spermary, ovary, Tiroidina, Parathyroid, lymphsystem and marrow all is very suitable object.
These cells both can be the cells of directly gathering from this biological tissue, also can be the cells of separating from the movement of biologies such as urinating, noise made in coughing or vomiting is eaten, can be again the culturing cells of succeeding transfer culture.The information of expecting relevant cancer is preferably used the cell of gathering from tumor tissues.
The character of the mammalian cell of identifying comprises the propagation energy and the grade of malignancy of tested cell.The propagation of so-called cell can phalangeal cell the proliferation activity level, be that the relevant controlling organization of whether giving propagation brings and (has or not canceration) unusually and the information of aspect such as dysploidy.The grade of malignancy of so-called cell particularly just is meant easy metastasis degree, easily recurs degree and prognosis mala degree etc.
At this, after recurrence was meant in order to extract malignant tumour and partly excises internal organs, same malignant tumour appearred in remaining internal organs once more; And tumour cell is separated to tissue (internal organs at a distance) at a distance, the propagation (transfer and relapse) of supporting oneself there from former focus.Made a definite diagnosis be considered as " the easily recurrence " of recurrence in general 5 years.If by classification by stages, III phase recurrence rate is 50%, than easily recurrence of II phase (recurrence rate 20%).So-called prognosis refers to predict advancing of disease process and final result, and the mortality ratio after 5 years or 10 years is high more, and prognosis is bad more, is 50% such as III phase mortality ratio, than II phase (mortality ratio 20%) poor prognosis.
So-called cyclin-dependent kinase is and the plain general name that combines the enzyme group who is activated of cycle, plays a role according to the specific period of different sorts in the cell cycle.So-called CDK inhibitor is to be attached in the plain CDK mixture of cycle to suppress its active factor group general name.
At this, the so-called cell cycle is that cell is from beginning propagation, get back to the loop cycle of starting point through processes such as dna replication dna, karyomit(e) distribution, nuclear fission, cytokinesises to splitting into two daughter cells, it is divided into G1 phase, S phase, G2 phase and M phase four-stage as shown in Figure 1.The S phase is duplicating period of DNA, and the M phase is division stage.The G1 phase is for some time that is accomplished to the synthetic beginning of DNA from mitotic division, is that the phase is checked in the preparation that enters the M phase.One crosses the stagnation point (being the R point) of G1 phase in zooblast, the cell cycle promptly starts, and carries out a week without a break in the way usually.The G2 phase is the DNA end of synthesis to for some time that mitotic division begins.The main test point of cell cycle be from the G1 phase enter the S phase eve and from the G2 phase to mitotic ingress.Particularly G1 phase check position is most important for inducing the S phase to begin.Because certain of a mistake G1 phase a bit, cell also can be bred without a break even without proliferation signal, and S → G2 → M → G1 carries out the cell cycle.In addition, stopping proliferating cells having a resting stage (G0) that keeps the dna content of G1 phase, is in the state that breaks away from the cell cycle, enters the S phase after slightly long some time of the G1 phase under the effect of proliferation-inducing in the comparable cell cycle.
The employed cyclin-dependent kinase of method of the present invention (CDK) preferably is selected from the biological group that is made of CDK1, CDK2, CDK4, CDK6, plain A dependant kinase of cycle, plain B dependant kinase of cycle and plain D dependant kinase of cycle.Plain A dependant kinase of so-called cycle refers to combine the active CDK of demonstration with plain A of cycle, present known have CDK1 and CDK2.Plain B dependant kinase of so-called cycle refers to combine the active CDK of demonstration with plain B of cycle, the present known CDK1 that has.Plain D dependant kinase of so-called cycle refers to combine the active CDK of demonstration with plain D of cycle, present known have CDK4 and CDK6.
So far as is known, these CDK are as shown in table 1, are combined into plain CDK mixture of cycle (following claim sometimes " active form CDK ") with each self-corresponding cycle element, activate the specific period of listed cell cycle of table 1.For example, plain A of CDK1 and cycle or B, CDK2 and plain A of cycle or E, CDK4 and CDK6 with the cycle element D1, D2, D3 combine generation active form CDK.On the other hand, the activity of CDK is subjected to the inhibition of the listed CDK inhibitor of table 1 sometimes.For example, P21 suppresses CDK1,2, and P27 suppresses CDK2,4,6, and P16 suppresses CDK4,6.
Table 1
| CDK | Bonded cycle element | Bonded CDK inhibitor | The action period of active form CDK |
| CDK4 CDK6 | Plain D3 of plain D2 cycle in plain D1 cycle in cycle | P27、P16 | G1 |
| CDK2 | Plain E of cycle | P27 | G1→S |
| CDK2 | Plain A of cycle | P21、P27 | Activate the S phase |
| CDK1 | Plain B of plain A cycle in cycle | P21 | G2→M |
| Plain A dependant kinase of cycle | Plain A of cycle | P21、P27 | CDK1:G2 → interim phase of M CDK2:S |
| Plain B dependant kinase of cycle | Plain B of cycle | P21 | CDK1：G2→M |
| Plain D dependant kinase of cycle | Plain D of cycle | P27、P16 | CDK4、6：G1 |
In above-mentioned CDK, measure expression amount and the activity value of CDK more than two kinds, ask in various CDK their ratio (the CDK specific activity of representing in the formula promptly or its inverse number), can draw the CDK profile.
CDK specific activity=CDK activity value/CDK expression amount
Therefore, the CDK profile particularly, such as profile (CDK specific activity inverse number profile) that the profile (CDK specific activity profile) that contains the CDK specific activity is arranged and contain CDK specific activity inverse number etc.
So-called CDK activity value refers to make how many substrates (such as active form CDK1, active form CDK2 is histone 1 with plain combination of specific period, active form CDK4 and active form CDK6 are the level (unit represents with U) of this kinase activity of Rb (retinoblastoma protein) phosphorylation, can measure with known always enzyme assay method.Particularly, can from the cell solution of measuring sample, prepare the sample that contains active form CDK, use
32P mark ATP (γ-(
32P)-ATP) will
32P puts into the labelled amount that the substrate protein white matter is measured the phosphorylated substrate that is labeled, and the typical curve of drawing with standard substance is that benchmark carries out quantitatively.As the method for not using the radioactive substance mark, go up disclosed method open 2002-335997 number if any Japanese Patent.This method is, the sample that contains targeted activity type CDK from the cell solution preparation of measuring sample, make gland glucoside 5 '-O-(3-tri o cresyl thiophosphate cresols ester) (ATP-γ S) and substrate reactions, the Serine or the threonine residues that monobasic thiophosphoric acid base are added this substrate protein white matter, allow the sulphur atom in the monobasic thiophosphoric acid base of mark fluorescent material or marker enzyme and adding combine, the labelled amount of the thiophosphoric acid base that mensuration is labeled (with the mark fluorescent material time be fluorescence volume), in addition quantitative according to the typical curve of drawing with standard substance.
Prepare by gathering target CDK specifically for measuring active sample from the solution of tested cell, also can prepare target CDK with the anti-CDK antibody of specificity, therefore, the plain dependant kinase of mensuration specific period (such as: plain A dependant kinase of cycle, plain B dependant kinase of cycle and plain E dependant kinase of cycle) time, available anti-CDK Antibody Preparation target CDK.Whatsoever all can there be active form CDK CDK in addition to be contained in the sample under the situation.Combine the mixture that forms such as also containing the CDK inhibitor with plain CDK mixture of cycle.Using under the situation of anti-CDK antibody, can comprise the mixture etc. of mixture, CDK and other compounds of CDK monomer, CDK and cycle element and/or CDK inhibitor.Therefore, activity value is that the substrate unit (U) as phosphorylation is measured under the state that active form, nonactive type, various competing reaction are mixed in.
So-called CDK expression amount is the target CDK amount of measuring from cell solution (corresponding to the unit of molecule number), can be by the well-known method mensuration of measuring the target protein quality from protein mixture.Such as, can use enzyme-linked immunosorbent assay method (ELISA method) and immunoblotting (Westernblot), also can measure with patent disclosure 2003-130871 disclosed method.Target protein (CDK) is caught with specific antibody and is got final product.Such as, can capture with anti-CDK1 antibody and to be present in intracellular CDK1 all (mixture that comprises mixture, CDK and other compounds of CDK monomer, CDK and cycle element and/or CDK inhibitor).
Therefore, we can say that the specific activity that calculates with above-mentioned formula is equivalent to show active CDK ratio among the intracellular CDK, is the CDK activity level based on the vegetative state of being identified zooblast.The CDK specific activity of so trying to achieve does not rely on the mensuration preparation method of sample.Measure preparation method of sample, particularly from the mensuration sample (cell solution) of biopsy material preparation be subject to contained acellular sex organization the tissue of actual acquisition, such as the influence of the number of extracellular substrate, therefore use specific activity or its meaning of its inverse number of having eliminated this influence very big, simple activity value compared with the past is higher with clinically property correlation.
Contain the CDK specific activity more than two kinds or the CDK profile of its inverse number by understanding, just know which CDK activity is more active, thereby the cell proportion of learning which bigger for the cell proportion in what or which period in period.
Be the center with the CDK specific activity profile that contains the CDK specific activity below, method of the present invention is described.
The kind of measuring the CDK of specific activity needs only according to the kind of inquisitive character suitably to be selected.Cancer cells is often escaped a large amount of propagation of normal propagation control, therefore, can imagine at the cell proportion of S phase and G2 phase higherly, can think just in canceration this moment.This cancer be we can say soon to pernicious.Also have, dysploidy be it is generally acknowledged through the unusual M phase or directly enter S through the G1 phase without the M phase and take place during the phase, therefore is present in the cell proportion of M phase and also can be described as pernicious less.Therefore, using CDK1 to be plain dependant kinase of period 1, is plain dependant kinase second cycle with CDK2, hives off by the size of CDK1 specific activity, and in having the group of similar CDK1 specific activity, the value of CDK2 specific activity is the value of reflection S phase cells ratio.Cell in the S phase is many, then can judge organizing clinically to pernicious of this cellularity, promptly easily shifts, the malignant cancer of prognosis mala.
In addition, also can be on CDK kind and the known action according to the CDK specific activity profile that contains CDK specific activity more than two kinds infer the cell cycle specific period have ratio, a grade of malignancy of identification of cell; Can also obtain with the related normal tissue cell in advance is the CDK specific activity profile more than two kinds that standard cell lines is measured, by relatively identifying grade of malignancy with normal cell.
As CDK specific activity profile, preferably adopt the ratio of the specific activity of two kinds of cyclin-dependent kinases.At this moment, compare the character of identification of cell by ratio certain threshold value corresponding with this ratio with the specific activity of two kinds of cyclin-dependent kinases.
The threshold value of using in the cellularity authentication method of the present invention is suitably set according to tested cell category and certified variety.As long as the setting of threshold value is from the database of the CDK specific activity of the numerous cells relevant with institute survey project, individual database and this cell, selects for this project to get final product as the value of the ratio of the specific activity of threshold value.Such as, about the cancer grade of malignancy, the tumour cell that the diagnosis of pathologist is often gathered with regard to known several patients is earlier on one's body obtained the ratio of two kinds of CDK specific activitys that are considered to dependency respectively, then with the ratio of trying to achieve by descending series arrangement, get just in time and arrangement can be divided into binary intermediate value as threshold value.
Method of the present invention be not only applicable to cell propagation can and character such as grade of malignancy, also be applicable to the susceptibility of cell to excitor substance.Result from the susceptibility difference of cell such as, the difference of the anticancer drug effect that pointed in recent years patient's genetic constitution causes to excitor substance.That is, whether responsive the character that cell had originally also comprise excitor substance this character, and the ratio of two kinds of CDK specific activitys is also relevant with the susceptibility to excitor substance.Therefore, the susceptibility authentication method of mammalian cell of the present invention is to identify that according to the expression amount of the 2nd CDK when that comprises the expression amount of a CDK and activity value and the CDK profile of the ratio of activity value above-mentioned cell is to the susceptibility of excitor substance.The profile (CDK specific activity inverse number profile) that above-mentioned CDK profile has the profile (CDK specific activity profile) that contains the CDK specific activity and contains CDK specific activity inverse number.The mode of using CDK specific activity profile such as have by with the specific activity of plain dependant kinase of period 1 with second cycle plain dependant kinase the ratio of specific activity and set threshold value compare and identify the susceptibility of this cell excitor substance.
Excitor substance is selected from multiplicaiton factor, anticarcinogen and variation originality material.Identify that disconnected content is different because of the kind of excitor substance with the susceptibility content.Such as, to anticarcinogen, identifying has inefficacy, and variation originality material and multiplicaiton factor are identified that no response etc. is arranged.
Threshold value as standard of perfection in cellular sensitivity authentication method of the present invention is suitably set according to the kind of excitor substance.Whether responsive the setting of threshold value be to investigate the active ratio of original CDK as the cell identification of indicator and relevant these cells correlation of data to excitor substance in advance, the selection sensitivity and insensitive between value as threshold value.Threshold value both can have been divided the reality patient group of haptic stimutus material this stimuli sensitive group and non-sensitive group in pairs, set according to its relation with the ratio of two kinds of CDK specific activitys measuring separately; Also can stimulate the culturing cell of various tumour cells earlier with stimulator, measure its propagation level and, it is divided into responsive group and non-sensitive group, set according to itself and relation of the ratio of two kinds of CDK specific activitys of mensuration separately to the reaction level of stimulator.
According to the kind of tested cell or the kind of stimulator, occur this situation sometimes: the CDK specific activity of one or both that are used to identify itself is too small or excessive on the contrary, and active ratio and threshold value are differed greatly.Sometimes also can occur being accredited as in the non-sensitive cell of this stimulator as the too small situation of the active ratio of CDK of tested project itself.In this case,, preferably also consider the size of the active ratio of CDK that is used to identify, identify the susceptibility of stimulator not only only according to the judgement recently of active ratio.Particularly, also want setting threshold as identification of indicator, adopt comparative result during evaluation with this threshold value for active ratio itself.
If when the susceptibility of identifying stimulator, with the ratio of active ratio of CDK and CDK specific activity respectively, be not particularly limited for its comparative sequence, but preferably suitably accept or reject according to different stimulator kind and cell category as index and threshold ratio.Such as, can be earlier with active ratio and threshold ratio, to clearly judging responsive or after insensitive part identifies, comparing by ratio corresponding threshold with regard to remaining cell again and make judgement with specific activity.Or when one or both specific activity of tested cell when too small or one or both active ratio is excessive, also threshold ratio that can be corresponding with the ratio of CDK specific activity earlier, after whether identifying sensitivity, only too small with regard to one or both specific activity again or the cell that one or both active ratio is excessive and the threshold value of this activity ratio compare, again whether responsively identify, revise the qualification result that ratio and respective threshold comparative result according to specific activity draw with this.
In addition, active ratio because of the measuring method of activity and expression amount and the antibody type that uses in measuring etc. different.Therefore, in authentication method of the present invention with threshold value relatively serve as the method identified of basis, the specific activity measuring method of making in the data base procedure when requiring setting threshold is necessary more consistent than measuring method with tested cell activity.
Embodiment
At first, the measuring method with regard to the mensuration method of sample preparation used among the following embodiment and CDK specific activity, CDK expression amount describes.
[measuring the preparation of sample]
As biological material to be determined, use be each tissue (2mm that surgery is gathered
3) or the culturing cell of the listed tumour of table 2.
Table 2
| Culturing cell | The tumour kind |
| KATOIII | Cancer of the stomach |
| K562 | Leukemia |
| Colo205 | Large bowel cancer |
| HeLa | Cervical cancer |
| MCF-7 | Mammary cancer |
| SW-480 | Large bowel cancer |
| SKBr | Mammary cancer |
| T47D | Mammary cancer |
In the dissolving damping fluid that contains 0.1w/v% Nonidet P40 (NP-40) (calbiochem corporate system), 50mM Tutofusin tris (TRIS) hydrochloric acid (pH7.4), 5mM ethylene dinitrilotetra-acetic acid (EDTA), 50mM Sodium Fluoride, 1mM sodium vanadate and 100 μ l/ml protease inhibitor cocktails (SIGMA corporate system), in ice bath, 5ml syringe with band 23G syringe needle aspirates biological material 10 times repeatedly, the preparation cell solution.Culturing cell then is formulated as 1 * 10
7Cell/5ml.
Down centrifugal removed insoluble matter 5 minutes with 15000rpm speed at 4 ℃, with supernatant liquor (cell solution) as the mensuration sample.
[the active mensuration of CDK]
From the sample of above-mentioned preparation, extract in the Ai Bende pipe of a certain amount of adding 1.5ml, its amount is that the whole protein quality that contains 100 μ g solutes in the 500 μ l dissolving damping fluid gets final product, as sample.
Adding in above-mentioned sample has specific antibody (anti-CDK1 antibody of polyclone of Santa Cruz (Santa Cruz) bio-engineering corporation or the anti-CDK2 antibody of polyclone) 2 μ g and albumin A sepharose (Bole (Bio-Rad) corporate system) 20 μ l after reacting 1 hour under 4 ℃ of environment to CDK to be measured, clean three times with damping fluid (0.1%NP-40,50mM Tutofusin tris hydrochloric acid, pH7.0), outstanding turbid in 15 μ l enzyme buffer liquid again, obtain containing target CDK sample bonded particulate sample.
All CDK monomers of this sample, cycle are plain to be caught in conjunction with the active form CDK, the active form CDK that form and CDK inhibitor complexes and CDK and CDK inhibitor complexes when not distinguishing (following be referred to as " CDK group "), and combines with particle.Measure this CDK group's activity with following method.
Preparation contains histone H1 (Upstate corporate system) 10 μ g, thio triphosphates adenosine (ATP-γ S, SIGMA corporate system) 5mM as CDK1 and CDK2 substrate and the end liquid of damping fluid (20mM Tutofusin tris hydrochloric acid (pH7.0), 0.1% polyoxyethylene (9.5)-Te Xin phenol (TritonX-100)), this end liquid is added above-mentioned CDK sample to 50 μ l, vibration is 10 minutes in 37 ℃ of environment, is cultivated.Be shown below, substrate Serine or threonine residues are produced the monothio phosphorylated substrate by active form CDK phosphorylation.
[Chemical formula 1]
After the reaction, the speed centrifugal treating with 1000rpm makes solids precipitation, the supernatant liquor 30 μ l of draw dissolved monothio phosphoric acid.In this supernatant liquor 18 μ l, add the binding buffer liquid 15 μ l that contain 150mM Tutofusin tris hydrochloric acid, pH9.2 and 5mM EDTA, be placed on the dark place incubated at room temperature 90 minutes again in 10mM iodacetyl luciferin solution (100mM Tutofusin tris hydrochloric acid (pH7.5), 1mM EDTA), so soon the sulphur in the thiophosphoric acid of monothio phosphorylated substrate is made fluorescent mark.By adding the reaction that the 6-mercaptoethanol stops iodacetyl fluorescein and thiophosphoric acid.
To be added on the pvdf membrane by fluorescently-labeled thiophosphoric acid substrate 0.4 μ g and inhale and move with strip trace (slotblotter).With the film of gained with 1% bovine serum albumin (BSA) sealing 30 minutes, under 37 ℃, allow antibiosis protein FITC (Vector system) react 1 hour, use 50mM TBS (25mM Tutofusin tris hydrochloric acid (pH7.4), 150mM sodium-chlor (NaCl)) to clean then three times 10 minutes.Use the image on fluorescent image analyzer (Bole's corporate system) analyzing film after cleaning.Activity is tried to achieve according to typical curve.
Typical curve forms by measuring active drafting of CDK with only containing the contained targeted activity type of K-562 chronic myeloid leukemia cells solution CDK, that concentration is different.
The same active enzyme amount of enzymic activity (the enzyme amount that a certain amount of matrix is changed) when therefore, the active 1U of tested CDK is for demonstration and K-562 total cellular protein 1 μ g.
[mensuration of CDK expression amount]
To soak initialized poly(vinylidene fluoride) (PVDF) film (Mi Libo (MILLIPORE) corporate system) through TBS (25mM Tutofusin tris hydrochloric acid (pH7.4), 150mM sodium-chlor (NaCl)) earlier and be installed on the strip trace (slotblotter), in each pond of strip trace (slotblotter) (2 * 2 * 3mm, permission capacity 100 μ l), respectively inject the cell solution for preparing above the 50 μ l.Each pond internal protein total amount respectively contains 5～15 μ g.
After the injection, from the bottom in pond, be that inhaled about 15 seconds with the negative pressure of about 200mmHg at the back side of film, sample is inhaled be imprinted on the film.
Next, to inject each pond with the solution of anti-CDK1 antibody of sample specificity bonded rabbit or the anti-CDK2 antibody of rabbit (antibody), after at room temperature placing about 30 minutes, inhaled about 15 seconds with the negative pressure of 500mmHg from the bottom, pond, use TBS (25mM Tutofusin tris hydrochloric acid (pH7.4), 150mM sodium-chlor (NaCl)) to clean again.
Inject each 40 μ l of FITC mark suis antibiosis protein reagent respectively, put at room temperature about 30 minutes, the secondary antibodies of promptly having used the FITC mark.Inhaled about 15 seconds with the negative pressure of 500mmHg from the bottom, pond, use TBS (25mM Tutofusin tris hydrochloric acid (pH7.4), 150mM sodium-chlor (NaCl)) to clean again.
Pvdf membrane is removed from base plate, clean, in 20% methyl alcohol, soaked 5 minutes with distilled water.Drying at room temperature is after about 15 minutes then, analyze, measure the proteinic fluorescence intensity that is adsorbed on the film with image analyzer (Bole company), according to the ready-formed typical curve, quantitatively (with weight (mg) the conversion amount corresponding with the CDK number of standard protein) is by the protein of FITC mark (CDK1 or CDK2).The CDK that determines like this measures the total amount for CDK group's (mixture of mixture, CDK and other compounds of CDK monomer, CDK and cycle element and/or CDK inhibitor etc.) of being contained in the cell.
Typical curve is to make like this: the pure product recombinant C DK protein soln that injects 5 kinds of concentration of 50 μ l in the TBS that contains 0.005%NP-40 and 50 μ g/ml BSA to each pond of the above-mentioned same processing of process respectively, make the FITC mark with above-mentioned same method, measure fluorescence intensity, the relation of fluorescence intensity and amount is showed.
[calculating of CDK specific activity]
From the CDK activity of aforesaid method mensuration and the measured value of CDK expression amount, calculate CDK specific activity (mU/ng or U/ng) with following formula.
CDK specific activity=CDK activity value/CDK expression amount
[embodiment 1: the character of various cancer cells]
In order to understand the pathology character of various cancer cells, listed culturing cell dna content of table 2 and CDK specific activity have been investigated.
(1) dna content
Target cell with after trypsinase/EDTA processing separates, is cleaned two times with PBS,, reclaim cell (2 * 10 through 100 * g, 4 ℃, 5 minutes centrifugal treating
5～1 * 10
6Cell).The cell limit of reclaiming is stirred with vortex stirrer, and the limit slowly adds in advance the 70% ethanol 1ml that is cooled to-20 ℃, fixes, and reacts more than 2 hours at 4 ℃ or-20 ℃ again.Behind two times cells of PBS cleaning, add RNase (SIGMA corporate system), 50 μ g/ml iodate third ingots (PI), the 1mg/ml glucose/PBS of 20KU/ml, cell is fully disperseed, room temperature reaction 1 hour is given the DNA fluorescent dye with PI.After the dyeing, remove impurity, measure and resolve and encouraged the fluorescence that wavelength is 488/536nm, 617nm with flow cytometer (FACScalibur (BD company)) with 35mm nylon mesh (FALCON).
Fig. 2 is each measurement result, and ordinate zou is a fluorescence intensity in Fig. 2, is equivalent to cell count, and X-coordinate is a dna content.
The dna content of zooblast and the relation of cell count generally as shown in Figure 3, with the cell cycle interlock.According to the measurement result of Fig. 2, with the ratio that exists of the cell of Modfit cell cycle analysis software (design of U.S. Verity Software House company) investigation S phase and G1 phase, its result as shown in Figure 4.
About KATOIII cell, K562 and Colo205 cell, from Fig. 2 and Fig. 4 as can be seen the S phase have the ratio height, about KATOIII cell, Colo205 cell and HeLa cell from Fig. 2 and Fig. 4 dysploidy (the particularly chromosome number of aneuploid) as can be seen
(2) CDK specific activity
About the listed culturing cell of table 2,, obtain its specific activity separately to measuring activity, the expression amount of CDK1 and CDK2 with the mensuration sample of specimen preparation method preparation according to the said determination method with said determination.The result as shown in Figure 5.
Thin black line is the specific activity of CDK2 among Fig. 5, and thick white line is the specific activity of CDK1, and left ordinate zou is the specific activity of CDK1, and right ordinate zou is the specific activity of CDK2.
The size of attention CDK1 specific activity can be known, roughly can be divided into the CDK1 specific activity than low group (KATOIII, K-562, Colo205, HeLa and MCF-7 cell) with than high group (SW480, SKBr3, T47D).And in the middle of each group, how much corresponding the size of CDK2 specific activity is with the cell proportion of measuring the dna content gained S phase.That is, only with the size of CDK2 specific activity can't make its with the S phase exist ratio corresponding, have only after hiving off according to the active ratio of CDK1 earlier could with the size of CDK2 specific activity and cell is pernicious to connect.
[embodiment 2: pathologist is to the diagnosis of biopsy sample and the relation of CDK specific activity profile]
Investigated the relation of postoperative distant metastasis type recurrence in 5 years at reality from the biopsy sample of cancerous tissue sampling with the CDK specific activity profile of depicting according to the CDK1 specific activity and the CDK2 specific activity of aforesaid method mensuration.
(1) diagnosis of pathologist
At the biopsy sample of actual 77 patients with mastocarcinoma (No.1～77), the diagnostic result of pathologist (TNM classification, nodus lymphoideus transferring rate state, cancerous tissue size, postoperative had or not recurrence and recurrence position in 5 years) is listed as table 3 and table 4.77 people all are breast carcinoma of early stage (I phase or the IIA phase).
In the table 3, the nodus lymphoideus transferring rate state during " LN " expression operation; Lymphoglandula is not made a definite diagnosis transferrer under " a " expression; Lymphoglandula is made a definite diagnosis and is shifted 1～3 person under " b " expression; Lymphoglandula is not made a definite diagnosis and is shifted 4 above persons under " c " expression.The size of original focus during " T " expression operation, diameter of tumor 2cm is following to be " a ", and diameter of tumor 2cm～5cm is " b ", and diameter of tumor 5cm is above to be " c ".
(2) threshold setting
Gather the biopsy sample from 126 patients with mastocarcinoma, with aforesaid method formation determination sample, measure CDK1 specific activity and CDK2 specific activity, CDK2 specific activity/CDK1 the specific activity of the cancer group (126 cancer patientss' cancerous tissues) that measures is arranged in order from low beginning, the value that group can be into 63 to 63 be as than threshold value.Threshold value is 46, is diagnosed as recurrence rate low (low dangerous) with what the ratio of CDK2 specific activity/CDK1 specific activity was lower than threshold value (46), is diagnosed as recurrence rate height (high-risk) with what the ratio of CDK2 specific activity/CDK1 specific activity was higher than threshold value (46).
(3) according to CDK specific activity profile diagnosis grade of malignancy
With the biopsy sample of gathering on one's body from 77 patients formation determination sample according to the method described above, measure CDK1 specific activity and CDK2 specific activity, be divided into high-risk group and low dangerous group according to (2) preset threshold for the cancer group (77 cancer patientss' cancerous tissues) that measures.The diagnostic result of making according to the specific activity profile and the diagnostic result of pathologist together see Table 3 tables 4.
Being ordinate zou when drawing CDK specific activity skeleton diagram, be that the about 46 times scale of CDK specific activity is drawn the skeleton diagram that shows CDK specific activity profile result with respect to each patient's (X-coordinate) CDK1 specific activity and CDK2 specific activity with the scale of CDK2 specific activity.Be diagnosed as low dangerous group patient No.1～42 CDK profile result as shown in Figure 6, be diagnosed as high-risk populations patient No.43～77 CDK profile result as shown in Figure 7.
Table 3
| Patient No. | TNM | LM | T | Recurrence | The recurrence position | The grade of malignancy diagnosis |
| 1 | I | a | a | Do not have | - | Low dangerous |
| 2 | I | a | a | Do not have | - | Low dangerous |
| 3 | I | a | a | Do not have | - | Low dangerous |
| 4 | I | a | a | Do not have | - | Low dangerous |
| 5 | I | a | a | Do not have | - | Low dangerous |
| 6 | I | a | a | Do not have | - | Low dangerous |
| 7 | I | a | a | Do not have | - | Low dangerous |
| 8 | I | a | a | Do not have | - | Low dangerous |
| 9 | I | a | a | Do not have | - | Low dangerous |
| 10 | I | a | a | Do not have | - | Low dangerous |
| 11 | I | a | a | Do not have | - | Low dangerous |
| 12 | I | a | a | Do not have | - | Low dangerous |
| 13 | I | a | a | Do not have | - | Low dangerous |
| 14 | IIA | a | b | Do not have | - | Low dangerous |
| 15 | IIA | a | b | Do not have | - | Low dangerous |
| 16 | IIA | a | b | Do not have | - | Low dangerous |
| 17 | IIA | a | b | Do not have | - | Low dangerous |
| 18 | IIA | a | b | Do not have | - | Low dangerous |
| 19 | IIA | a | b | Do not have | - | Low dangerous |
| 20 | IIA | a | b | Do not have | - | Low dangerous |
| 21 | IIA | a | b | Do not have | - | Low dangerous |
| 22 | IIA | a | b | Do not have | - | Low dangerous |
| 23 | IIA | a | b | Do not have | - | Low dangerous |
| 24 | IIA | a | b | Do not have | - | Low dangerous |
| 25 | IIA | a | b | Do not have | - | Low dangerous |
| 26 | IIA | a | b | Do not have | - | Low dangerous |
| 27 | IIA | a | b | Do not have | - | Low dangerous |
| 28 | IIA | a | b | Do not have | - | Low dangerous |
| 29 | IIA | a | b | Do not have | - | Low dangerous |
| 30 | IIA | a | b | Do not have | - | Low dangerous |
| 31 | IIA | a | b | Do not have | - | Low dangerous |
| 32 | IIA | a | b | Do not have | - | Low dangerous |
| 33 | IIA | a | b | Do not have | - | Low dangerous |
| 34 | IIA | a | b | Do not have | - | Low dangerous |
| 35 | IIA | a | b | Do not have | - | Low dangerous |
| 36 | IIA | a | b | Do not have | - | Low dangerous |
| 37 | IIA | a | b | Do not have | - | Low dangerous |
| 38 | IIA | a | b | Do not have | - | Low dangerous |
| 39 | IIA | a | b | Do not have | - | Low dangerous |
| 40 | IIA | a | b | Do not have | - | Low dangerous |
[table 4]
| Patient No. | TNM | LM | T | Recurrence | The recurrence position | The grade of malignancy diagnosis |
| 41 | IIA | b | a | Do not have | - | Low dangerous |
| 42 | IIA | b | a | Do not have | - | Low dangerous |
| 43 | I | a | a | Do not have | - | High-risk |
| 44 | I | a | a | Do not have | - | High-risk |
| 45 | I | a | a | Do not have | - | High-risk |
| 46 | IIA | a | b | Do not have | - | High-risk |
| 47 | IIA | a | b | Do not have | - | High-risk |
| 48 | IIA | a | b | Do not have | - | High-risk |
| 49 | IIA | a | b | Do not have | - | High-risk |
| 50 | IIA | a | b | Do not have | - | High-risk |
| 51 | IIA | a | b | Do not have | - | High-risk |
| 52 | IIA | a | b | Do not have | - | High-risk |
| 53 | IIA | a | b | Do not have | - | High-risk |
| 54 | IIA | a | b | Do not have | - | High-risk |
| 55 | IIA | b | a | Do not have | - | High-risk |
| 56 | IIA | a | b | Do not have | - | High-risk |
| 57 | IIA | b | a | Do not have | - | High-risk |
| 58 | IIA | a | b | Do not have | - | High-risk |
| 59 | IIA | b | a | Do not have | - | High-risk |
| 60 | IIA | b | a | Do not have | - | High-risk |
| 61 | IIA | b | a | Do not have | - | High-risk |
| 62 | IIA | b | a | Do not have | - | High-risk |
| 63 | IIA | b | a | Do not have | - | High-risk |
| 64 | IIA | b | a | Do not have | - | High-risk |
| 65 | IIA | b | a | Do not have | - | High-risk |
| 66 | I | a | a | Have | Skin | High-risk |
| 67 | I | a | a | Have | Lung | High-risk |
| 68 | IIA | a | b | Have | Skin | High-risk |
| 69 | IIA | a | b | Have | Lymphoglandula | High-risk |
| 70 | IIA | a | b | Have | Liver, bone | High-risk |
| 71 | IIA | a | b | Have | Pleura | High-risk |
| 72 | IIA | a | b | Have | Lung | High-risk |
| 73 | IIA | a | b | Have | Bone | High-risk |
| 74 | IIA | a | b | Have | Skin | High-risk |
| 75 | IIA | a | b | Have | Bone | High-risk |
| 76 | IIA | a | b | Have | Lymphoglandula | High-risk |
| 77 | IIA | a | b | Have | Lung | High-risk |
As table 4, Fig. 6, shown in Figure 7, being considered to has 35 examples (No.43～77) to be accredited as high-risk at CDK specific activity profile in not poor early-stage breast cancer patient 77 examples of grade of malignancy, wherein 12 examples (No.66～77) postoperative recurrence in 5 years.On the other hand, being accredited as the recurrence rate of hanging down in the 42 dangerous examples at CDK specific activity profile is zero.Hence one can see that, if not only compare active ratio of CDK1 and the active ratio of CDK2, consider that also the ratio (in the present embodiment for CDK2 specific activity/CDK1 specific activity) of the two draws the profile of organizing that comes from patients with mastocarcinoma, then the grade of malignancy height of cancer when the low and CDK2 specific activity of CDK1 specific activity is high.On the contrary, the grade of malignancy of cancer is low when the low and CDK1 specific activity of CDK2 specific activity is high.In other words, even we can say CDK2 specific activity same high (such as No.38 and No.67), the cancer grade of malignancy that the CDK1 specific activity is high relatively is low.
More specifically can with default certain threshold, from comparative result, identify the character and the grade of malignancy of cancer.
[embodiment 3: to the susceptibility of anticarcinogen polyenoid taxol]
(1) setting of threshold value and standard of perfection
Before accepting cancer drug therapy, gather breast carcinoma tissue from 1000 patients with mastocarcinoma accepting taxanes (taxane) pharmacological agent, prepare the mensuration sample as stated above with these breast carcinoma tissue of gathering, measure its activity and expression amount with regard to CDK1 and CDK2 respectively, calculate specific activity.On the other hand, these patients with mastocarcinoma are carried out taxanes (taxane) pharmacological agent, its result is divided into the situation that situation that tumour dwindles and tumour are not dwindled, sets CDK2 specific activity/CDK1 specific activity (threshold value of ratio), make it the tumour size is divided into and dwindle group and the non-group of dwindling.At this moment, the ratio threshold value of specific activity is 16.
Extreme when big or extreme hour setting threshold at the active ratio of CDK1 or the active ratio of CDK2 again, dwindle group and the non-group of dwindling so that can be categorized as tumor size according to the active ratio of CDK1 or the active ratio of CDK2.At this moment, the threshold value of the active ratio of CDK1 is 20, and the threshold value of the active ratio of CDK2 is 500 and 10000.
Followingly make evaluation: at first, with the value of CDK1 specific activity and CDK2 specific activity and threshold ratio, the active ratio of CDK1 not to 20 (the active ratio of CDK1＜20) and the active ratio of CDK2 be accredited as non-sensibility during not to 500 (the active ratio of CDK2＜500), range the non-sensibility prediction group.On the other hand,, then be diagnosed as susceptibility, range the susceptibility prediction group if the active ratio of CDK2 surpasses 10000 (the active ratio of CDK2＞10000).
Then, to in above-mentioned evaluation, neither belonging to the crowd that the non-sensibility prediction group does not belong to the susceptibility prediction group again, CDK2 specific activity/CDK1 specific activity and threshold value (16) are compared, and this ratio is that 16 above persons are accredited as the susceptibility crowd, is accredited as the non-sensibility crowd less than 16.
In addition, in the present embodiment, the active ratio of CDK1 is that 0 (the active ratio of CDK1=0) and the active ratio of CDK2 are 500 or more when (500≤CDK2 activity ratio≤10000) below 10000, for simplicity, CDK2 specific activity/CDK1 specific activity is considered as infinity, promptly the ratio of specific activity surpasses threshold value 16 at this moment, is diagnosed as susceptibility.
(2) CDK specific activity profile and susceptibility prediction
As stated above prepare mensuration sample with the breast carcinoma tissue that the patients with mastocarcinoma A～I that never carries out anticancer drug therapy gathers on one's body, measure its active and CDK expression amount with regard to CDK1 and CDK2 respectively, calculate specific activity.
(ratio of A～CDK1 I) and the value of CDK2 specific activity and CDK2 specific activity/CDK1 specific activity relatively is categorized as susceptibility group and non-sensibility group with (1) middle preset threshold according to 9 patients with mastocarcinoma.Again patient A～I (X-coordinate) is produced the demonstration CDK1 activity CDK specific activity profile of CDK2 specific activity (ordinate zou) relation when, as Fig. 8.At this, the ordinate zou ratio of CDK2 specific activity is made as 16 times of CDK1 specific activity ordinate zou ratio.
According to above-mentioned threshold value, patient A～I is categorized as polyenoid taxol susceptibility prediction group and non-sensibility prediction group, the result is as follows:
Polyenoid taxol non-sensibility prediction group: patient A, B, C
Polyenoid taxol susceptibility prediction group: patient D, E, F, G, H, I
(3) curative effect of anticarcinogen polyenoid taxol
Above-mentioned patients with mastocarcinoma A～I is treated with anticarcinogen polyenoid taxol (Aventis corporate system), and dose is each 60mg/m
2(body surface area), every 3～4 weeks once, amount to four times.Whether the tumour size dwindles after the investigation medication.
Whether tumour dwindles, by the palpation of pathologist, according to Therasse P, ArbuckSG, Eisenhauer EA, Wanders J, Kaplan RS, Rubinstein L, Verweij J, Van GlabbekeM, van Oosterom AT, Christian MC, andGwyther SG:New guidelines to evaluate theresponse to treatment in solid tu mors.J.Natl.Cancer Inst 2000; The standard of perfection of the last record of 92:205-216, the result dwindles for the tumour that is judged as of " fully take a turn for the better " and " part takes a turn for the better ", the result be " stubborn disease " and " PD " be judged as that tumour is non-dwindles.
Be not judged as the patient that tumour is dwindled: A, B, C, D
Be judged as the patient that tumour is dwindled: E, F, G, H, I
9 philtrums have 8 people with predict the outcome consistent, (positive prediction rate) 100% unanimity when confirming actual efficacy particularly.
[embodiment 4: to the susceptibility of anticarcinogen taxol]
(1) setting of threshold value and Case definition
Quote in embodiment 3 preset threshold and standard of perfection.
(2) CDK specific activity profile and susceptibility prediction
As stated above prepare mensuration sample with the breast carcinoma tissue that the patients with mastocarcinoma J～P that never carries out anticancer drug therapy gathers on one's body, measure its active and CDK expression amount with regard to CDK1 and CDK2 respectively, calculate specific activity.
(ratio of J～CDK1 P) and the value of CDK2 specific activity and CDK2 specific activity/CDK1 specific activity relatively is categorized as susceptibility group and non-sensibility group with (1) middle preset threshold according to 7 patients with mastocarcinoma.Again patient J～P (X-coordinate) is produced the demonstration CDK1 activity CDK specific activity profile of CDK2 specific activity (ordinate zou) relation when, as Fig. 9.At this, the ordinate zou ratio of CDK2 specific activity is made as 16 times of CDK1 specific activity ordinate zou ratio.
According to above-mentioned threshold value, patient J～P is categorized as taxol susceptibility prediction group and non-sensibility prediction group, the result is as follows:
Taxol non-sensibility prediction group: patient J, K
Taxol susceptibility prediction group: patient L, M, N, O, P
(3) curative effect of anticarcinogen taxol
Above-mentioned patients with mastocarcinoma J～P is treated with anticarcinogen taxol (Bristol drugmaker system), and dose is each 80mg/m
2(body surface area), weekly, whether continuous use 12 all " Invest, Then Investigate " tumor size dwindle.According to judging with polyenoid taxol same standard whether tumor size dwindles.
Judged result is as follows:
The patient that tumour is not dwindled: J
The patient that tumour is dwindled: K, L, M, N, O, P
8 philtrums have the 7 people consistent (accuracy: 87.5%), be accredited as male patient 100% with method of the present invention and confirm that tumour dwindles of judged result with actual anticarcinogen curative effect that predicts the outcome.
Industrial utilizability:
The cells involved character that authentication method according to the present invention draws particularly qualification result and the clinician of cancer grade of malignancy has very high correlation at the on-the-spot diagnostic result of doing of medical treatment, therefore, can be used as the means of making a definite diagnosis of the disease that the cell proliferation regulative mechanism obstacles such as cancer are caused. Not only can be used for the research to people's disease, can also be used for the research to the cell proliferation regulative mechanism obstacle of various mammalian cells.
Zooblast of the present invention can be used as investigation cell proliferation to the sensitiveness authentication method of excitor substance and has or not the method for impact to be used on the various medicaments such as anticarcinogen, MF, variation originality material and medicine. Particularly can be used for judging the different curative effects of the medicaments such as anticarcinogen that different curative effects and the individual difference of the medicaments such as anticarcinogen of different carcinoma cell cause. Can predict before actual medication with the method whether the selection anticancer drug therapy is effective, therefore, sensitiveness authentication method of the present invention can be utilized as qualified Therapeutic selection index.
Claims (17)
1. mammalian cell character authentication method, it is characterized in that:, the character of above-mentioned cell is identified according to the activity value that comprises plain dependant kinase of mammalian cell period 1 (CDK) and the activity value of element dependant kinase second cycle when (the 2nd CDK) of expression amount and the CDK profile of the ratio of expression amount.
2. the described authentication method of claim 1, it is characterized in that: the CDK profile by more described CDK profile and standard cell lines is identified above-mentioned character.
3. mammalian cell character authentication method, it is characterized in that:, identify above-mentioned mammalian cell character by comparing with the ratio of the activity value of the ratio (A1) of the activity value of plain dependant kinase of mammalian cell period 1 (CDK) and expression amount and plain dependant kinase second cycle (the 2nd CDK) and the ratio (A2) of expression amount and corresponding to certain threshold value of this ratio.
4. a certain the authentication method of describing in the claim 1～3, it is characterized in that: described cyclin-dependent kinase is selected from the biological group that is made of CDK1, CDK2, CDK4, CDK6, plain A dependant kinase of cycle, plain B dependant kinase of cycle and plain D dependant kinase of cycle.
5. the authentication method described of claim 4, it is characterized in that: the described period 1, plain dependant kinase was CDK1, second cycle, plain dependant kinase was CDK2.
6. a certain the authentication method of describing in the claim 1～5, it is characterized in that: described character is the propagation energy of mammalian cell.
7. a certain the authentication method of describing in the claim 1～6, it is characterized in that: described character is the grade of malignancy of mammalian cell.
8. the authentication method described of claim 7, it is characterized in that: described grade of malignancy is easy metastatic rate.
9. the authentication method described of claim 7 is characterized in that: described grade of malignancy is easy recurrence degree.
10. the authentication method described of claim 7, it is characterized in that: described grade of malignancy is the prognosis mala degree.
11. the authentication method that claim 8 is described, it is characterized in that: described transfer is nodus lymphoideus transferring rate and distant metastasis.
12. one kind according to a certain the authentication method of describing carries out the diagnosis of cancer grade of malignancy to the cell that picks up from biological tissue Method for cancer diagnostics in the claim 1～11.
13. mammalian cell susceptibility authentication method, it is characterized in that:, above-mentioned cell is identified the susceptibility of excitor substance according to the activity value that comprises plain dependant kinase of mammalian cell period 1 (CDK) and the activity value of element dependant kinase second cycle when (the 2nd CDK) of expression amount and the CDK profile of the ratio of expression amount.
14. mammalian cell susceptibility authentication method, it is characterized in that:, judge the susceptibility of above-mentioned cell excitor substance by comparing with the ratio of the activity value of the ratio (A1) of the activity value of plain dependant kinase of mammalian cell period 1 (CDK) and expression amount and plain dependant kinase second cycle (the 2nd CDK) and the ratio (A2) of expression amount and corresponding to certain threshold value of this ratio.
15. claim 13 or the 14 susceptibility authentication methods of describing, it is characterized in that: the activity value of plain dependant kinase of period 1 (CDK) and the ratio (A1) of expression amount and/or the activity value of plain dependant kinase second cycle (the 2nd CDK) and the ratio (A2) of expression amount are compared with each self-corresponding certain threshold value respectively, identify the susceptibility of above-mentioned mammalian cell excitor substance.
16. a certain authentication method of describing in the claim 13～15 is characterized in that: described cyclin-dependent kinase is selected from the biological group that is made of CDK1, CDK2, CDK4, CDK6, plain A dependant kinase of cycle, plain B dependant kinase of cycle and plain D dependant kinase of cycle.
17. a certain authentication method of describing in the claim 13～16 is characterized in that: described excitor substance is selected from multiplicaiton factor, anticarcinogen and variation originality material.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP160389/2004 | 2004-05-31 | ||
| JP2004160389 | 2004-05-31 | ||
| JP375639/2004 | 2004-12-27 |
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| CN1957088A true CN1957088A (en) | 2007-05-02 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200580016823 Pending CN1957088A (en) | 2004-05-31 | 2005-05-30 | Method of judging properties of mammalian cell and method of diagnosing cancer |
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| Country | Link |
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| CN (1) | CN1957088A (en) |
| ES (1) | ES2354616T3 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101402992A (en) * | 2007-10-02 | 2009-04-08 | 希森美康株式会社 | Device for supporting diagnosis of a cancer and a device for predicting an effects of anthracycline anticancer drugs |
| CN105242034A (en) * | 2011-07-22 | 2016-01-13 | 希森美康株式会社 | Method for providing oncogenesis information and device for providing oncogenesis information |
-
2005
- 2005-05-30 CN CN 200580016823 patent/CN1957088A/en active Pending
- 2005-05-30 ES ES05743830T patent/ES2354616T3/en active Active
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101402992A (en) * | 2007-10-02 | 2009-04-08 | 希森美康株式会社 | Device for supporting diagnosis of a cancer and a device for predicting an effects of anthracycline anticancer drugs |
| CN105242034A (en) * | 2011-07-22 | 2016-01-13 | 希森美康株式会社 | Method for providing oncogenesis information and device for providing oncogenesis information |
| CN105242034B (en) * | 2011-07-22 | 2019-02-05 | 希森美康株式会社 | Cancerous information providing method and cancerous information provide device |
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| Publication number | Publication date |
|---|---|
| ES2354616T3 (en) | 2011-03-16 |
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