WO2004003564A2

WO2004003564A2 - Tumour marker and the use thereof for the diagnosis and treatment of tumour diseases

Info

Publication number: WO2004003564A2
Application number: PCT/EP2003/006748
Authority: WO
Inventors: Stephanie Lamer; Marie-Laure Fogeron; Hermann Lage; Udo Kellner
Original assignee: Europroteome Ag
Priority date: 2002-06-26
Filing date: 2003-06-26
Publication date: 2004-01-08
Also published as: WO2004003564A3; AU2003246592A1

Abstract

The invention relates to the use of the type II membrane protein NP 055070 (respectively Swiss-Prot number), NHP2-type protein 1 P55769, pre-mRNA cleavage factor Im (25kD) NP008937, Lysyl-tRNA synthetase Q15046, UNR-interacting protein Q9Y3F4, nucleic transport factor 2 P13662, erythrocyte phosphatase1 isoenzyme F P24666, Prefoldin sub-unit 2 Q9UHV9, heterogeneous nuclear ribonucleoprotein C1/C2 P07910, transitional endoplasmic reticulum-ATPase P55072, bifunctional methylenetetrahydrofolic acid dehydrogenase P13995, 47 kDa heat-shock protein precursor P29043, ubiquinol-cytochrome C reductase complex core protein P31930, the fragments thereof, detector substances targeting the same, and/or nucleic acids coding for the same, for producing a means for diagnosing tumours and for the prophylactic or therapeutic treatment of tumours.

Description

Tumor markers and their use for the diagnosis and therapy of tumor diseases

The invention relates to the use of markers for the detection of tumors and for the therapy of tumor diseases. The following proteins could be identified as a means for cancer risk assessment, diagnosis and therapy of tumors or carcinomas: Type II membrane protein NP 055070 (each Swiss Prot. Number), NHP2-like protein 1 P55769, pre-mRNA cleavage factor Im (25kD) NP008937, lysyl-tRNA synthetase Q15046, UNR-interacting protein Q9Y3F4, nuclear transport factor 2 P13662, erytrocyte phosphatase isoenzyme F P24666, prefoldin subunit 2 Q9UHV9, heterogeneous nuclear ribonucleofluidic acid p2, p2-oligonucleotide protein-C1, p2-oligonucleotide protein-C1, p2-oligonucleotide protein-C1, p2-oligonucleotide protein-C1, p2-oligonucleotide-C1-P3 P13995, 47 kDa heat shock protein precursor P29043, ubiquinol-cytochrome C reductase complex core protein P31930, its fragments, recognition substances directed against these and / or nucleic acids coding for them.

A tumor is a locally defined increase in tissue volume, although in the broader sense any localized swelling due to edema, acute or chronic inflammation or the like can be referred to as a tumor. In the narrower sense, however, only new tissue formations, such as blastoma or neoplasia, are understood in the form of spontaneous, differently uninhibited, autonomous and irreversible excess growth of the body's own tissue as a tumor, whereby the tumor growth is associated with a loss of specific cell and tissue functions can according to her biological behavior, according to a histogenetic system or according to clinical and pathological findings.

In the clinical area, it is necessary to detect tumors as early and selectively as possible, because early detection and subsequent removal ensures that the tumor can be removed successfully without the affected organs being deformed too much and metastases forming. Even in the case of follow-up examinations after cancer surgery, the smallest metastases must be detected early in order to optimize further follow-up treatment. For some areas of occupational medicine, it is still necessary to determine whether a tissue or organ shows a potential susceptibility to cancer without being degenerate or transformed.

The easiest way to identify a tumor is to feel and look. For example, breast cancer can be felt as a lump in the breast. Indications of skin cancer can be visually recognized by the doctor through conspicuous birthmarks. Other optical methods are the imaging methods. With the help of apparatus, images of the body are recorded on which a tumor can be identified. These methods include, for example: X-ray and computer tomography (CT). In both methods, the body is X-rayed, whereby the degenerated tissue structures can be seen. With the help of CT, three-dimensional images can also be recorded. So-called contrast agents are often used in these methods, which are injected into the corresponding regions and increase the absorption, which makes tumors more recognizable. Cancer diagnosis using ultrasound and the use of radioactively labeled antibodies is also possible. This recognize tumor-typical antigens on the organs to be examined, bind there and can thus make tumors recognizable.

In addition to imaging methods, laboratory tests are an important means of early cancer detection. Samples of urine, blood and also tissue samples are examined for abnormalities. On the one hand, this can be a changed composition, but also the occurrence of substances that normally do not occur or only occur in small quantities. These substances are called tumor markers. They are either produced by the tumor tissue itself or as a reaction of the body to the tumor. In addition to substances, tumor markers also refer to cellular changes, the qualitative or quantitative analysis of which enables a statement to be made about the presence, the course or a prognosis of malignant diseases. Tumor markers are usually physiologically occurring substances that can be detected in increased or decreased concentrations compared to physiological conditions in serum, urine or other body fluids, whereby these substances are synthesized and secreted in the tumor tissue and released by tumor decay or formed as a reaction of the organism to a tumor. A large number of tumor markers are known: the prostate-specific antigen (PSA) and the prostatic acid phosphatase occur in prostate carcinomas. Alpha tetoprotein is a protein that is normally produced by the fetus. If it is found in the blood of non-pregnant women or men, it indicates a liver or germline tumor. Lactate dehydrogenase (LDH) is an important enzyme in the human body and is found in almost every tissue. A greatly increased level of LDH in the blood can be caused by forms of cancer such as leukemia. In over 50% of all cancer cases there is a mutation in the p53 gene, which can lead to the formation of p53 autoantibodies, which are indicative transformed cells can be detected. Cellular tumor markers are, for example, hormone receptors, receptors for growth-promoting substances in leukemia and cell markers, which indicate an increased expression of 5 oncogenic genes and monoclonal cell growth.

A disadvantage of the known methods for the detection of tumors is that all methods, regardless of whether the doctor e.g. the prostate carcinoma by palpation

10 detected or whether specific antigens are used for detection on the tumor surface, make a tumor recognizable only when the tumor has reached a critical size. That is, early stages of tumor growth can be achieved using known methods and means

15 Tumor diagnostics including the use of tumor markers cannot be determined.

Cancer diagnosis using tumor markers has further disadvantages. In this way, tumor markers can also appear in non-carcinogenic diseases; continues to mean a

20 No presence or no evidence of tumor markers does not indicate that there is no tumor disease. Another disadvantage is that the tumor markers are usually non-specific. This means that positive proof of the nature of the

25 shows tumor disease. A further disadvantage is that the known means and methods cannot be used to determine whether a tissue that is currently still healthy and unremarkable can degenerate at a later point in time, that is to say whether it tends to develop from

30. Controlled to switch to uncontrolled cell growth. Another, very decisive disadvantage of the known methods of early tumor detection is that they are not used to monitor the development of tumors, for example after an operation in which certain tumors have been removed can be used. A feature of all known methods for early tumor detection, in particular when using tumor markers, is that they can only diagnose a very narrow range of tumors, so that a certain amount of false positive and false negative results must be expected.

The object of the invention was therefore to provide means and methods for tumor diagnosis and therapy which do not have the disadvantages mentioned and in particular allow simple, safe and inexpensive detection or therapy of tumor diseases.

The present invention solves this technical problem by using type II membrane protein NP 055070 (Swiss-Prot. Number), NHP2-like protein 1 P55769, pre-mRNA cleavage factor Im (25kD) NP008937, lysyl-tRNA synthetase Q15046, UNR -interagierendes protein Q9Y3F4, nuclear transport factor 2 P13662, Erytrozytenphosphatasel isoenzymes F P24666, Prefoldin subunit 2 Q9UHV9, heterogeneous nuclear ribonucleoprotein C1 / C2 P07910, transitionale endoplasmic reticulum ATPase P55072, bifunctional Methylenetetrahydrofolsäure dehydrogenase P13995, 47 kDa heat shock protein precursor P29043, ubiquinol-cytochrome C Reductase complex core protein P31930, its fragments, recognition substances directed against these and / or nucleic acids coding for them for the production of an agent for the diagnosis, prophylactic or therapeutic treatment of tumors.

The proteins mentioned belong functionally or structurally to closely related protein families. The tumor markers advantageously make it possible to diagnose cancer at a very early stage and also to make statements about the future degeneration or transformation of certain tissues. Furthermore, the tumor markers are used to monitor the development of tumors. For the purposes of the invention, the proteins mentioned also include polypeptides which are at least 80%, at least 90% and particularly at least 95%, 97% or 99% homologous to these. The invention also relates to proteins modified by inversions, deletions, insertions and additions, provided that at least some of the essential functions of the wild-type proteins are present. It is also possible for natural amino acids to be replaced by synthetic amino acids in the proteins. The proteins mentioned therefore concern not only the naturally occurring proteins but also all modifications, mutants or derivatives, such as proteins produced by means of recombination techniques. Such a protein may also include unusual amino acids and / or modifications such as alkylation, oxidation, thiol modification, denaturation and oligomerization and the like.

A sample in the sense of the invention is the designation for a biological good taken by sampling or a part or a small amount of such, the nature of which is to be tested chemically, biologically, clinically or similarly. The sampling is carried out from the patient or from acquired humoral or cellular components of the patient in particular such that the extracted subset ^• an average of the entire quantity. The characteristics determined by examining the sample are used to assess the amount recorded by the sample, which allows conclusions to be drawn about the total amount, for example an entire organ, such as the liver, spleen, blood or the immune system. For the examination, the samples can be pretreated by mixing, dividing, crushing, adding enzymes or markers or otherwise. Various possibilities for the pretreatment of samples are known to the person skilled in the art. For the purposes of the invention, nucleic acids are nucleic acids which either encode the proteins, the recognition substances or their fragments. Of course it is possible that the nucleic acids are also hybridizing nucleic acids.

In the context of the invention, recognition substances are molecules which can interact with the proteins mentioned, the nucleic acids encoding them or their fragments in such a way that the proteins and / or the nucleic acids can be detected. The recognition substances can in particular be specific proteins which bind the proteins mentioned, antibodies, fluorescent markers, labeled carbohydrates or lipids, antisense constructs, cDNA or mRNA molecules, their fragments or the like. Of course, it is also possible that the recognition substances do not detect the proteins, but rather antibodies directed against them. In this case, the recognition substances would be, for example, secondary antibodies. The invention also relates to a method for the detection of tumors in the biological sample of a patient, wherein in the sample a level of at least one of the proteins selected from the group consisting of type II membrane protein NP 055070 (each Swiss Prot. Number), NHP2- similar protein 1 P55769, pre-mRNA cleavage factor Im (25kD) NP008937, lysyl-tRNA synthetase Q15046, UNR-interacting protein Q9Y3F4, nuclear transport factor 2 P13662, erytrocyte phosphatase isoenzyme F P24666, prefoldin nucleate9 Q9 riboninogen unit9 C19 , transitional endoplasmic reticulum ATPase P55072, bifunctional methylenetetrahydrofolic acid dehydrogenase P13995, 47 kDa heat shock protein precursor P29043, ubiquinol-cytochrome C reductase complex core protein P31930, its fragments, detection substances directed against these and / or nucleic acids coding for them are compared with a control level of a control sample from a healthy patient and the tumor is detected on the basis of the modified level in the sample in comparison with the control level. Healthy in the sense of the invention does not have to mean the complete absence of diseases or pathogenic changes. The healthy patient represents either a single patient or an average number of patients who can serve as a comparison group in such a way that a change in the level of the tumor markers mentioned can be determined. A modification of the level compared to the control level means that the tumor markers mentioned have changes in their concentration or activity as protein, as nucleic acid or as antibodies compared to a control level.

In a particular embodiment of the invention, the level is determined as a protein concentration, a protein activity, a concentration of isoforms, a DNA, an RNA concentration, a gene expression and / or a copy number of a nucleic acid which codes for one of the proteins. The person skilled in the art can advantageously choose various options for determining the level of the tumor markers. One possibility, for example, is to determine the protein concentration using spectrographic methods. However, it is also possible to determine the level at the RNA or DNA level or, for example, via the activity of the individual proteins. It is of course also possible that the tumor marker is only present in the tumor or is absent there.

In a further preferred embodiment of the invention, the sample is brought into contact with a recognition substance of at least one of the proteins mentioned and the binding of the recognition substance to the protein is determined. Recognition substances which can be used with advantage are, for example, antibodies which are directed against the protein or antisense constructs which can bind the proteins themselves or the nucleic acids which code for these proteins. The antibodies, the antisense constructs or other molecules to be used as the recognition substance can be labeled or not labeled, it being possible, for example, to label them with fluorescent labels. However, it is also advantageously possible to use non-labeled substances. Various possibilities are known to the person skilled in the art to determine the interaction of recognition substances and proteins or the nucleic acids coding for them. However, it is also possible for the proteins to be determined on the basis of the antibodies which are directed against them. Here, a second antibody, a so-called secondary antibody, would bind the antibody that originally bound the protein and thus detect it.

The invention also relates to a method for the treatment of tumors, the level of the proteins, the recognition substances and / or the nucleic acids being modified. Various possibilities are known to the person skilled in the art for modifying the level of the substances or molecules mentioned. For example, the level of proteins can be increased or decreased. An increase is possible, for example, by adding an additional promoter to the nucleic acid encoding the corresponding protein or by increasing the activity of the original promoter. It is also possible to increase the number of copies of the nucleic acids in the corresponding target tissue, for example a tumor tissue, as a result of which more proteins are expressed. However, the level of the proteins mentioned can also be reduced. It is possible to lower the level of the proteins by using antisense constructs the nucleic acids that code for the proteins interact, are added to the corresponding tumor tissue, as a result of which the nucleic acids can no longer be read sufficiently. However, it is also possible to allow antibodies directed against the proteins to interact with them, so that the concentration of the proteins does not change, but their activity is suppressed.

In a preferred embodiment of the invention, the level of NHP2-like protein 1 P55769, pre-mRNA cleavage factor Im (25kD) NP008937, lysyl-tRNA synthetase Q15046, UNR-interacting protein Q9Y3F4, heterogeneous nuclear ribonucleoprotein C1 / C2 P07910, transitional endoplasmic reticulum ATPase P55072, 47 kDa heat shock protein precursor P29043 and / or ubiquinol-cytochrome C reductase complex core protein P31930 reduced.

In a further preferred embodiment of the invention, the level of type II II membrane protein NP 055070, nuclear transport factor 2 P13662, erytrocyte phosphate isoenzyme F P24666, prefoldin subunit 2 Q9UHV9 and / or bifunctional methylenetetrahydrofolic acid dehydrogenase is increased in the treatment of tumors.

The invention also relates to an agent for the diagnosis and / or therapy of tumor diseases, which comprises the proteins mentioned, recognition substances and the nucleic acids.

The invention also relates to a kit for the detection of tumor cells, the kit comprising at least one of the proteins mentioned, the nucleic acids and / or the recognition substances. The invention is to be illustrated in more detail below using an exemplary embodiment, without restricting it.

Example 2D protein gel electrophoresis

In order to obtain information about differentially expressed proteins, the cell lines were treated with or without a cytostatic agent for 24 h. The sensitive parental cells were treated with the dose of 20 ng / ml mitoxantrone or 250 ng / ml daunorubicin that was therapeutically achieved in the patient's serum. The resistant cells, however, were treated with the 10-fold higher concentration for the same period. The cells grew in 20 cm O petri dishes and were detached in ice-cold PBS-EDTA (10 mM EDTA); adhering cells were additionally scraped off. According to Bradford [Bradford 1976], the protein was determined before lysis. After centrifugation at 500 g, the cell pellet was placed directly in 8.3 M urea,

2 M thiourea, 100 mM DTT, 4% CHAPS, 2% Servalyt 4-9 (Serva) and a spatula tip bromophenol blue lysed.

100 μg of total protein was applied to gel strips (pH

3 - 10; Amersham Pharmacia Biotech) and focused overnight at 50 V (total 80 kVh at 20 C °). The molecular weight was separated in the second dimension. For this, the strips were incubated twice for 15 min in 50 mM Tris pH 8.8 with 6 M urea, 2% SDS, 30% glycerol and 1% DTT and in the Ettan DALT II apparatus (Amersham Pharmacia Biotech) at 2.5 W. / Gel for 30 min, followed by 19 W / gel for 6 h, separated [Shevchenko, Wilm et al. 1996]. The commercially cast gels had a polyacrylamide concentration of T = 12.5% with a cross-linking of C = 3%. The silver color of the gels was carried out as described by Gharahdaghi, Weinberg et al. 1999 described.

Figure 7: Example of the proteome gel evaluation. In the case of protein gel after cytostatic treatment, the gray levels of the spots were replaced by corresponding green values and overlaid with 50% transparency over the red-colored gel image to be compared. Overlapping spots that produced a gray value are under both conditions to be compared

equally expressed, while shimmering green are overexpressed and shimmering red are underexpressed (labeled U in the figure). Gel Evaluation

The gels were then scanned and evaluated in the computer using the Adobe Photoshop 5.5 computer program, in which the images were first made semi-transparent. A direct comparison of two silver gels was achieved by replacing black with red in one picture and black with green in the other picture. After superimposing these two digitally modified images, spots of the same size produced a shade of gray (proteins expressed equally strongly), whereas green or red spots indicated over- or under-expression (Figure 7). The evaluation was carried out in small areas in which the appropriate spots could be placed exactly one above the other. ace spectrometric analysis (ALDITOF)

Highly differently expressed proteins were analyzed by mass spectrometry. For this, the corresponding protein spots were cut out of the gel. The cut-out protein spots were then included

Trypsin (Promega) digested (0.05μg trypsin per spot in 15μl

50mM ammonium hydrogen carbonate buffer). The

Protein extraction was done with the ZipTip system

(Millipore), was eluted with 4μl 50% acetonitrile 0.1% TFA (trifluoro-vinegar). Then 0.4μl sample with 0.4μl

Matrix (α-cyano-4-hydroxy cinnamic acid: 5mg / ml in 50%

Acetonitrile 50% H ₂ 0 0.3% TFA) mixed and 0.8μl on the

Carrier (MALDI target) applied.

The Voyager STR from Applied Biosystems (20kV; Grid Voltage: 70%; Delay 200ns; positive reflector mode, low mass gate 580 Da; 5 spectra of 100 shots were collected.

Proteo ics

At the proteome level, we were interested in the differential protein expression of the two resistant ones

Lines compared to the parental cells as well

Consequences of exposure to cytostatics per se. For this, the parenteral line was in each case therapeutic, sublethal

Doses of 20ng / ml mitoxantrone and 250ng / ml daunorubicin treated for 24h. This dose corresponds to the therapeutic one

Average serum level in the patient. Since the two resistant lines grow constantly under mitoxantrone (200ng / ml) and daunorubicin (2.5μg / ml), we were also interested in what happens when the respective cytostatic agent is removed for 24 hours. protein spots

overexpressed 37 89 47 57 60 51 underexpressed 25 30 20 6 62 61 specifically overexpressed 17 71 2 51 13 38 specifically underexpressed 16 23 16 3 32 48 examined overexpressed 13 20 11 3 13 14 examined underexpressed 8 7 0 0 14 14 examined specifically overexpressed 1 4 0 0 1 6 examined specifically underexpressed 0 2 0 0 1 3

Table 6: Overview of the amount of differentially expressed protein spots. Specifically overexpressed or underexpressed refers to the proteins that were only differentially expressed under the condition specified above. Abbreviations: Mito = Mitoxantron, Dauno = Daunorubicin, P = EPG85-257P, RN = EPG85-257RN, RDB = EPG85-257RDB.

The resulting seven 2D protein gels showed approximately the same protein spotting pattern. Approx. 2500 protein spots could be differentiated per gel. The evaluation showed small differences in the cell lines ± cytostatic, while the comparison between the resistant lines and the parental line showed significantly more differences. The comparisons each showed a partly balanced but mostly a ratio which was shifted in favor of overexpression: Parent cell responses to mitoxantrone

downregulation

The parental cells after mitoxantrone treatment had 25 visibly downregulated protein spots, 9 of which were differentially expressed under other conditions (see Figure 25 and Tables 6 & 7). Except for one spot, all proteins down-regulated under various conditions were examined in the MALDITOF. Five of these proteins are in a molecular weight range between 19 and 35 kDa (proteins 1-5). The two proteins 40 and 41 were in the molecular weight range between approx. 130 and 170 kDa, each of which is the acyl-CoA binding protein, which stands for a reduced fat metabolism and speaks for a reduced cell turnover (proliferation). Protein 6 is based on the molecular weight ladder at 83 kDa. The MALDITOF analysis showed that it is the beta-1 tubulin with a molecular weight of 49.8 kDa. This protein was overexpressed in the EPF85-257RDB after omitting the maintenance dose of daunorubicin. Unfortunately, MALDITOF protein determination of spots 31, 40 and 41 was not possible because the amount of protein was not sufficient. With the exception of spot 1, the remaining spots 2 - 5 were at an IP of pH 6.1. Protein 1 corresponds to type II membrane protein (20.7kDa, pH 4.8) and is also downregulated in parental cells after daunorubicin administration, while the removal of mitoxantrone or daunorubicin leads to overexpression in the correspondingly resistant cells. The protein spots 2 - 5 & 40, 41 show the same behavior.

overexpression

Under the same experimental conditions, 37 Protein spots were found that were up-regulated in the mitoxantrone-treated cells. 17 were found to be specific, i.e. only overexpressed under these conditions. 14 spots are also differentially expressed under other conditions (Figure 25, Table 7). The distribution of the two spot groups was between approx. 19 and 120 kDa and the isoelectric point of the proteins was shifted to the basic range up to pH 10. 9 protein spots were overexpressed only in the parental cells but under both mitoxantrone and daunorubicin conditions. The protein 38: 54 kDa nuclear RNA and DNA-binding protein was also overexpressed in the mitoxantrone-resistant and daunorubicin-resistant cells and in the EPG85-257RN cells with mitoxantron, interrasante not in the parent or daunorubicin treated daunorubicin resistant cells. The proteasome subunit α type 7 (spot 10) was overexpressed only in the parental mitoxantrone-treated cell. Due to a lack of signal, spots 7, 9, 14, 17, 20 and 21 could not be identified. The lysyl-tRNA synthetase was only used in the parental cells but in both cytostatics

(Spot 24), the proteasome ε chain precursor (Spot 13) and the pre-mRNA cut factor IM (Spot 11) can be found. The heterogeneous nuclear ribonucleoprotein AI (Spot 12) is overexpressed under all conditions. The protein with the spot number 8 (NHP2-like protein 1) is expressed in the parental with both cytostatics and in the resistant with mitoxantrone. Parent cell responses to daunorubicin

downregulation

After daunorubicin treatment, the parental cells showed 30 downregulated protein spots. Of these, 7 were also differentially expressed under other conditions (see Figure 25 and Table 7). As with mitoxantrone treatment, five of the proteins which are differentially down-regulated under other conditions lie in a molecular weight range between 19 and 35 kDa (proteins 1-5; see 4.1.1.1.). The proteins 40 and 41, which were also differentially expressed under different conditions, were in the molecular weight range between approx. 130 and 170 kDa (see 4.1.1.1). All proteins had an isoelectric value in the neutral or slightly acidic pH range, only 2 protein spots that were specifically down-regulated under these conditions were in the slightly basic range. As described above, spots 3 and 4 are each the acyl-CoA binding protein. One of the two spots that are specifically differentially expressed by means of MALDITOF is the ubiquitin conjugation enzyme E2-17 (spot 16). The other spot could not be because of keratin contamination. Keratin cannot be determined. overexpression

In contrast, 89 protein spots were found upregulated in the daunorubicin-treated cells under the same experimental conditions. 71 spots were only differentially expressed under these conditions, while 18 spots also showed a different expression under other conditions (Figure 26, Table 7). The distribution of the two spot groups was between approx. 20 and 120 kDa and the isoelectric point of the proteins was rather shifted to the basic range up to pH 10, with proteins also occurring in the acid range. 9 protein spots were overexpressed only in the parental cells but under both mitoxantrone and daunorubicin conditions. 4 spots that were specifically overexpressed for the test conditions were examined in MALDITOF: spot 18 corresponds to the RHO GDP dissociation inhibitor that was found to be overexpressed in a mitoxantrone-resistant fibrosarcoma cell line, while the others could not be determined for various reasons (Table 7). Spot 26 did not produce a database result, with ATP synthetase being a candidate.

Responses of mitoxantrone-resistant cells to mitoxantron absence overexpression in the untreated cells

If the maintenance dose of mitoxantrone is omitted from the mitoxantrone-resistant cells, 20 protein spots are visibly upregulated. Of these, 4 were also differentially expressed under other conditions (see Figure 27 and Table 7). 4 Proteins specific under these conditions are all with their IP at pH 4, while the others, e.g. T. were also upregulated under other conditions in the alkaline range. The molecular weight of the protein spots found is between approximately 37 and 100 kDa.

Overexpression in the treated cells

Under these conditions, 47 proteins are overexpressed in the treated cells. The protein spectrum ranges from 19 to 200 kDa and from acidic (near pH3) to basic (near pHIO) depending on the gel resolution. Interestingly, only 2 spots could be found that exclusively express differentially only under these conditions in the mitoxantrone-resistant cells (a basic protein with an IP at pH 10 and a molecular weight of approx. 55 kDa, and an acidic protein with an IP near 3.0 and a molecular weight at 120 kDa). All other spots found are also differentially expressed under other comparable conditions (45 spots). Striking and marked with stars in Figure 27 is a protein pattern of 34 spots widely scattered in the gel, which only occurs in resistant cells treated with mitoxantrone (vs. EPG85-257P and RN untreated). It is therefore a question of controllable resistance-associated proteins. MALDITOF was used to examine 11 protein spots that were also overexpressed under other conditions (Table 7). It is the previously described NHP2-like protein 1 (spot 8), the heterogeneous nuclear ribonucleoprotein AI (spots 12 & 23), the heterogeneous nuclear ribonucleoprotein C / C2 (spots 33 & 34), the transitional endoplasmic reticulum ATPase (spot 35), the 54kDa nuclear RNA and DNA binding protein (spot 38) and the glutathione S-transferase (spot 48). The glutathione S-transferase is up-regulated in the two resistant lines compared to the parental lines, and the expression in the mitoxantrone-resistant lines can be increased by adding mitoxantrone. For technical reasons, spots 17, 32 and 37 did not give an evaluable signal.

Reactions of Daunorubicin-Resistant Cells to Daunorubicin Absence

Overexpression in the untreated cells If the maintenance dose of daunorubicin is omitted from the daunorubicin-resistant cells, 6 protein spots are visibly upregulated. Half (3) of these were also differentially expressed under other conditions (see Figure 28 and Table 7). The molecular weight of the The protein spots found are between approximately 32 and 100 kDa. A protein spot (approx. 90 kDa, IP at 4-5) is only downregulated in daunorubicin-treated cells (or overexpressed in the untreated ones; EPG85-257P and RDB; marked with *).

Overexpression in the treated cells

Under these conditions, 57 proteins are overexpressed in the treated cells. The differential protein spectrum ranges from 19 to 120 kDa and from acidic (near pH3) to basic (near pHIO). Interestingly, only 6 spots could be found that were exclusively differentially expressed only under these conditions only in the Dauήorubicin-resistant cells. All other spots found are differentially expressed under other comparable conditions (51 spots). In contrast to the observation in mitoxantrone-treated cells, there is only one protein marked with an asterisk, which only occurs in cells treated with daunorubicin (EPG85-257P and RDB; IP around 5, approx. 35kDa). 3 protein spots were investigated using MALDITOF, which were also overexpressed under other conditions (Table 7). It is the already described beta-1 chain of tubulin (spot 7) and the heterogeneous nuclear ribonucleoprotein AI (spots 12 & 23). The beta-1 chain of tubulin is also differentially expressed in the two parental cells with mitoxantrone and daunorubicing.

Changes in mitoxantrone-resistant cells compared to parental down-regulation

The resistant cells showed recognizable down-regulated protein spots compared to the parental cells 62. Of these, 29 became differential under other conditions are therefore not specific for this resistance

(see Figure 29 and Table 7). The proteins found are in the molecular weight range of approximately 19-150 kDA and an IP of approximately 3.5-9. The proteins differentially expressed under other conditions are also at

Mostly clustered in the low molecular weight range

(Figure 29, left blot bottom left). Twelve proteins that were downregulated in the resistant cells are among the proteins that are differentially expressed under other conditions. These include spots 3 and 4, which each correspond to the acyl-CoA binding protein, type II membrane-binding protein (spot 1), epidermal fatty acid-binding protein (spot 28) and iso-enzyme F of erythrocyte acid phosphatase 1 (spot 29) , while spots 5, 40 and 41 each gave no evaluable signal. Spots 27 and 30 and 31 were expressed specifically under these conditions. These are the nuclear transport factor 2 (27) and the prefoldin subunit 2, while the spot 31 gave no result. overexpression

Under the same experimental conditions, 60 protein spots were found that were up-regulated in the mitoxantrone-resistant cells. 36 were found to be specific, i.e. only overexpressed under these conditions. 14 spots are also differentially expressed under other conditions (Figure 29, Table 7). The distribution of the proteins was between approx. 19 and 150 kDa and the isoelectric point was also evenly distributed. 14 protein spots were specifically overexpressed only in the resistant cells, while 46 were also differentially expressed under other conditions. Striking and marked with stars in Figure 29 is a protein pattern of 34 spots that are widely scattered in the gel, which are only found in resistant cells treated with oxantrone occurs (vs. EPG85-257P and RN untreated). It is therefore a question of regulatable resistance-associated proteins.

14 spots were examined, one of which expresses differentially in this comparison (spot 39). These are heterogeneous nuclear ribonucleoprotein A2 / B1. The proteins differentially expressed under other conditions are: the NHP2-like protein 1 (spot 8), the heterogeneous nuclear ribonucleoprotein AI (spots 12 & 23), the UNR-interacting protein (spot 25) the heterogeneous nuclear ribonucleoprotein C1 / C2 ( Spots 33, 34), the transitional endoplasmic reticulum ATPase (spot 35), the protein 54 kDa nuclear RNA and DNA binding protein (spot 38) and the glutathione S-transferase (spot 48). Spot 37 gave no signal. Specifically, the heterogeneous nuclear ribonucleoprotein A2 / B1 and the spot 36, which gave no signal, were expressed.

Changes in daunorubicin-resistant cells compared to parental down-regulation

The resistant cells had visibly downregulated protein spots compared to the parental cells 61. Of these, 13 were also differentially expressed under other conditions, so they are not specific for this resistance (see Figure 30 and Table 7). The proteins found are in the molecular weight range of approx. 19 - 150 kDA and an IP of approx. 3 - 10. The proteins differentially expressed under other conditions are largely clustered in the acid / neutral low molecular weight range (pH 19 - 35; Figure 30, left) Blot bottom left). The 11 proteins that were downregulated in the resistant cells are among the proteins that are differentially expressed under other conditions. These include the both spots 3 and 4, each corresponding to the acyl-CoA binding protein, the beta-1 chain of tubulin (spot 6), the ubiquitin conjugation enzyme E2-17kDa (spot 16), the epidermal fatty acid binding protein (spot 28) and the isozyme F of the erythrocyte acid phosphatase 1 (spot 29), while the spots 2, 5, 15, 31, 40 and 41 each gave no evaluable signal. Interestingly, the isozyme F of erythrocyte acid phosphatase 1 only occurs in both resistant cells compared to the parental cells (EPG85-257RN and RDB). The bifunctional methylenetetrahydrofolic acid dehydrogenase (spot 42), the mitochondral precursor of enoyl-CoA hydratase (spot 43) and the transaldolase (spot 44) were expressed specifically under these conditions. overexpression

Under the same experimental conditions, 51 protein spots were found that were up-regulated in the Daunorubicin-resistant cells. 38 were found to be specific, i.e. only overexpressed under these conditions. 13 spots are differentially expressed under other conditions (Figure 30, Table 7). The distribution of the proteins was between approx. 32 and 130 kDa and the isoelectric point was also evenly distributed. 14 protein spots were specifically overexpressed only in the resistant cells, while 46 were also differentially expressed under other conditions. In contrast to the numerous conspicuous protein spots marked with stars, which only occurred in resistant cells treated with mitoxantron (vs. EPG85-257P and RN treated), there is only one such spot also marked with an asterisk for daunorubicin (Figure 30). It is therefore only a regulatable, resistance-associated protein.

14 spots were examined, six of which were specific overexpressed in this comparison (spots 45 - 47 & 49 - 51). This is the Spot 45, behind which there are only 3 different proteins: the mitochondral precursor of the aspartate aminotransferase, the precursor of the collagen-binding protein 2 and the precursor of the 47kDa heat shock protein. This also includes Annexin I (Spot 46), the Eta type of protein kinase C (Spot 47), the Cor protein of Ubiquinol cytochrome C reductase (Spot 49) and the 54kDa nuclear RNA and DNA binding protein (Spots 50 & 51). The proteins differentially expressed under other conditions are: the heterogeneous nuclear ribonucleoprotein AI (spots 12 & 23), the UNR interacting protein (spot 25), the heterogeneous nuclear ribonucleoprotein C1 / C2 (spots 33, 34) and the protein 54 kDa nuclear RNA and DNA binding protein (spot 38) and the glutathione S-transferase (spot 48). Spots 7 & 32 gave no signal.

Claims

claims

1. Use of type II membrane protein NP 055070 (each Swiss Prot. Number), NHP2-like protein 1 P55769, pre-mRNA cleavage factor Im (25kD) NP008937, lysyl-tRNA synthetase Q15046, UNR-interacting protein Q9Y3F4, nuclear transport factor 2 P13662, Erytrozytenphosphatasel Isoenzyme F P24666, Prefoldin subunit 2 Q9UHV9, heterogeneous nuclear ribonucleoprotein C1 / C2 P07910, transitional endoplasmic reticulum ATPase P55072, bifunctional methylenetetrahydrofolic acid Dehydrogenase P3503503903-P3503-D3503-D3503-D3503-D3503-P3503-D3503-C5503-C5303 , against these recognition substances and / or coding nucleic acids for the production of an agent for the diagnosis, prophylactic or therapeutic treatment of tumors.

2. Method for the detection of tumors in a sample from a patient, characterized in that in the sample a level of at least one of the proteins selected from the group consisting of type II membrane protein NP 055070 (each Swiss Prot. Number), NHP2- Similar protein 1 P55769, pre-mRNA cleavage factor Im (25kD) NP008937, lysyl-tRNA synthetase Q15046, UNR-interacting protein Q9Y3F4, nuclear transport factor 2 P13662, erytrocyte phosphatase isoenzyme F P24666, prefoldin nucleate9 Q9 riboninogen unit9 C19 , transitional endoplasmic reticulum ATPase P55072, bifunctional methylenetetrahydrofolic acid Dehydrogenase P13995, 47 kDa heat shock protein precursor P29043, ubiquinol-cytochrome C reductase complex core protein P31930, whose fragments determined against these recognition substances and / or nucleic acids encoding them, compared this level with a control level of a control sample from a healthy patient and compared the tumor based on the modified level in the sample compared to the control level.

3. The method according to claim 2, characterized in that as the level a protein concentration, a protein activity, a concentration of isoforms, a DNA, an RNA concentration, a gene expression and / or a copy number of a nucleic acid which codes for one of the proteins be determined.

4. The method according to claim 2 or 3, characterized in that the sample is brought into contact with a recognition substance for at least one of the proteins and the binding of the recognition substance to the protein is determined.

5. A method for the treatment of tumors, characterized in that the level of the proteins, the recognition substances and / or the nucleic acids is modified according to one of claims 1 to 3.

6. The method according to the preceding claim, characterized in that the level of NHP2-like protein 1 P55769, pre-mRNA cleavage factor Im (25kD) NP008937, lysyl-tRNA synthetase Q15046, UNR-interacting protein Q9Y3F4, heterogeneous nuclear ribonucleoprotein C1 / C2 P07910, transitional endoplasmic reticulum ATPase P55072, 47 kDa heat shock protein precursor P29043, and / or ubiquinol-cytochrome C reductase complex core protein P31930 is reduced.

Method according to claim 5, characterized in that the level of type II membrane protein NP 055070, nuclear transport factor 2 P13662, erytrocyte phosphatase isoenzyme F P24666, prefoldin subunit 2 Q9UHV9, and / or bifunctional

Methylenetetrahydrofolic acid dehydrogenase P13995, is increased.

8. Agents for the diagnosis and / or therapy of tumor diseases comprising type II membrane protein NP 055070 (each Swiss Prot. Number), NHP2 -like protein 1 P55769, pre-mRNA cleavage factor Im (25kD) NP008937, lysyl-tRNA synthetase Q15046, UNR-interacting protein Q9Y3F4, nuclear transport factor 2 P13662, erytrocyte phosphate isoenzyme F P24666, prefoldin subunit 2 Q9UHV9, heterogeneous nuclear ribonucleoprotein C1 / C2 P07910, transitional endoplasmic reticulum acid ATPase P55072

Dehydrogenase P13995, 47 kDa heat shock protein precursor P29043, ubiquinol-cytochrome C reductase complex core protein P31930, its fragments, recognition substances directed against these and / or nucleic acids coding for them.

9. Kit for the detection of tumor cells, characterized in that it comprises at least one protein, a nucleic acid and / or a recognition substance according to one of claims 1 to 3.