WO1998045429A2

WO1998045429A2 - Polypeptides involved in the staurosporine induced apoptotic pathway

Info

Publication number: WO1998045429A2
Application number: PCT/EP1998/002157
Authority: WO
Inventors: Dominik Imfeld; Peter Fürst; Patrick Schindler; Walter MÄRKI
Original assignee: Novartis Ag; Novartis-Erfindungen Verwaltungsgesellschaft Mbh
Priority date: 1997-04-10
Filing date: 1998-04-14
Publication date: 1998-10-15
Also published as: AU7524898A; GB9707307D0; WO1998045429A3

Abstract

The present invention relates to polypeptides involved in the apoptotic pathway, to a process of isolating said polypeptides, to polypeptides obtainable by said process, to the use of said polypeptides as a marker or surrogate marker of a cellular condition achievable by application of staurosporine to a cell, as a diagnostic marker or surrogate marker for a disease responsive to an induction or inhibition of a cellular condition achievable by application of staurosporine to a cell and to pharmaceutical compositions comprising said polypeptides.

Description

POLYPEPTIDES INVOLVED IN THE STAUROSPORINE INDUCED APOPTOΗC PATHWAY

Apoptosis, which is the process of programmed cell death, has emerged as a key regulatory mechanism fundamental to the development and maintenance of tissue homeostasis. Whereas too much cell death can cause degenerative disorders, such as neurodegenerative diseases and neuropathies, too little cell death may lead to proiiferative disorders, such as cancer or autoimmune diseases. Apoptosis is a crucial process removing cells in tissue development, normal cell turnover, hormone induced tissue atrophy and in pathological processes such as T-cell depletion in AIDS, autoimmune diseases like rheumatoid arthritis, neuronal degeneration in Huntington's disease, Alzheimer's disease and epilepsy. Cells undergoing apoptosis generally display shrinkage, loss of cell-cell- contact, chromatin condensation, membrane blebbing and internucleosomal degradation of DNA into discrete DNA fragments of approximately 200bp length.

Several molecules affecting apoptosis have been described, which, in principle, may exert at least part of their pharmacological effects via induction or via prevention of apoptosis. PKC-mediated phosphorylation of numerous protein substrates is associated with a wide range of biological effects. Staurosporine, an alkaloid isolated from streptomyces cultures, induces apoptosis (Bertrand et al., Exp. Cell Res.211 (1994), 314 - 321). Staurosporine is commercially available, e.g. from Calbiochem, La Jolla CA, USA. However, the pathways that link an effector to the cell death remain largely unknown. Accordingly, there is a need to provide cellular compounds, in particular polypeptides, which play key roles in the apoptotic process. Thus, an object of the present invention is to provide polypeptides involved in the signal transduction pathways during the apoptotic process. The polypeptide may either induce or inhibit apoptosis or a cellular status related to apoptosis. Hereinafter, said polypeptides will be referred to as "polypeptides of the invention".

It is a further object of the present invention to provide a process for the isolation of polypeptides of the invention Yet a further object of the present invention relates to the use of the polypeptides of the invention as a marker or surrogate marker for apoptosis or a cellular condition related to apoptosis, as a diagnostic marker or surrogate marker for said condition, as a drug discovery target, or as a drug. Other objects of the present invention are to provide a DNA sequence coding for the polypeptides of the invention, to provide an oligonucleotide capable of hybridizing to such a DNA sequence or to a related RNA molecule, and to provide an antibody directed to said polypeptides.

Figure 1 shows the Nano-ESMS spectrum of tryptic peptide mixture derived from protein spot 2A.2B of the present invention (upper trace). Nano-ESMS spectrum of m/z 656.4 (lower trace).

Figure 2 shows the formation of peptidic b and v fragments.

Figure 3 shows the search results for the sequence tag approach of spot 2B, the covered sequence being underlined.

Figure 4 shows the search results for the sequence tag approach of spot 12B, the covered sequence being underlined.

Unexpectedly, it has now been found that by treating the HL 60 cell line with staurosporine to induce the apoptotic pathway in the cell line a number of polypeptides, previously not known to play a role in the apoptotic pathway can be isolated from the cell lysate. These polypeptides, can be identified and subsequently isolated by detecting their differential expression pattern or differential migration behavior using two dimensional electrophoresis

Accordingly, the present invention provides in a first embodiment a polypeptide selected from the group consisting of :

(1A.1B) a polypeptide having on a 2D-gel an apparent pi value of 6.4 and an apparent molecular weight of 22 kDa;

(2A.2B) a polypeptide having on a 2D-gel an apparent pi value of 6.8 and an apparent molecular weight of 22 kDa; (3A) a polypeptide having on a 2D-gel an apparent pi value of 7.6 and an apparent molecular weight of 26 kDa;

(4A) a polypeptide having on a 2D-gel an apparent pi value of 4.8 and an apparent molecular weight of >100 kDa;

(5A) a polypeptide having on a 2D-gel an apparent pi value of 6.2 and an apparent molecular weight of 39 kDa;

(6A) a polypeptide having on a 2D-gel an apparent pi value of 5.9 and an apparent molecular weight of 40 kDa;

(7A) a polypeptide having on a 2D-gel an apparent pi value of 5.8 and an apparent molecular weight of 27 kDa;

(8A) a polypeptide having on a 2D-gel an apparent pi value of 5.5 and an apparent molecular weight of 26 kDa;

(9A) a polypeptide having on a 2D-gel an apparent pi value of 5.2 and an apparent molecular weight of 35 kDa;

(1 OA, 6B) a polypeptide having on a 2D-gel an apparent pi value of 5.7 and an apparent molecular weight of 19 kDa;

(11 A) a polypeptide having on a 2D-gei an apparent pi value of 5.7 and an apparent molecular weight of 15 kDa;

(12A, 3B) a polypeptide having on a 2D-gel an apparent pi value of 5.1 and an apparent molecular weight of 5 kDa;

(13A) a polypeptide having on a 2D-gel an apparent pi value of 5.3 and an apparent molecular weight of 25 kDa;

(14A) a polypeptide having on a 2D-gel an apparent pi value of 4.0 and an apparent molecular weight of 22 kDa;

(15A) a polypeptide having on a 2D-gel an apparent pi value of 4.0 and an apparent molecular weight of 20 kDa;

(16A) a polypeptide having on a 2D-gel an apparent pi value of 7.2 and an apparent molecular weight of >90 kDa;

(17A) a polypeptide having on a 2D-gel an apparent pi value of 4.85 and an apparent molecular weight of >100 kDa;

(18A) a polypeptide having on a 2D-gel an apparent pi value of 4.9 and an apparent molecular weight of >100 kDa;

(4B) a polypeptide having on a 2D-gel an apparent pi value of 7.6 and an apparent molecular weight of 25.5 kDa; (5B) a polypeptide having on a 2D-gel an apparent pi value of 5.7 and an apparent molecular weight of 17 kDa;

(6B) a polypeptide having on a 2D-gel an apparent pi value of 5.7 and an apparent molecular weight of 19 kDa;

(7B) a polypeptide having on a 2D-gel an apparent pi value of 5.5 and an apparent molecular weight of 26 kDa;

(8B) a polypeptide having on a 2D-gel an apparent pi value of 5.3 and an apparent molecular weight of 26 kDa;

(9B) a polypeptide having on a 2D-gel an apparent pi value of 4.6 and an apparent molecular weight of 27 kDa;

(10B) a polypeptide having on a 2D-gel an apparent pi value of 5.5 and an apparent molecular weight of 29 kDa;

(11 B) a polypeptide having on a 2D-gel an apparent pi value of 5.3 and an apparent molecular weight of 33 kDa;

(12B) a polypeptide having on a 2D-gel an apparent pi value of 6.0 and an apparent molecular weight of 44 kDa;

(13B) a polypeptide having on a 2D-gel an apparent pi value of 7.2 and an apparent molecular weight of >70 kDa;

(14B) a polypeptide having on a 2D-gel an apparent pi value of 7.0 and an apparent molecular weight of >100 kDa;

(15B) a polypeptide having on a 2D-gel an apparent pi value of 4.7 and an apparent molecular weight of 115 kDa;

(16B) a polypeptide having on a 2D-gel an apparent pi value of ? and an apparent molecular weight of 45 kDa;

(17B) a polypeptide having on a 2D-gel an apparent pi value of 6.2 and an apparent molecular weight of 19 kDa;

(18B) a polypeptide having on a 2D-gel an apparent pi value of 7.5 and an apparent molecular weight of 25 kDa; and

(19B) a polypeptide having on a 2D-gel an apparent pi value of 7.9 and an apparent molecular weight of 26 kDa.

The invention further provides a process for isolating a polypeptide, comprising (a) induction of a cellular condition related to apoptosis in the HL 60 cell line, said cellular condition being achievable by culturing said HL 60 cell line with an appropriate final concentration of staurosporine until a cellular condition related to apoptosis is detectable upon monitoring said cellular condition, (b) identification of a polypeptide showing upon analysis by 2- dimensional polyacrylamide gel electrophoresis, being in one dimension an immobilized pH- gradient electrophoresis and in the other dimension SDS polyacrylamide gel electrophoresis, a differential expression pattern or a differential migration behavior compared with the non-induced cell line and (c) isolation of said thus identified polypeptide.

In a preferred embodiment of the process of the invention said culturing of the HL 60 cell line is performed for at least 3 hours, preferably for 3 to 18 hours, more preferably for 10 or 12 hours.

In a further preferred embodiment of said process said polypeptide shows a differential expression pattern, i.e. emergence of a signal corresponding to said polypeptide, disappearance of a signal corresponding to said polypeptide. In a further preferred embodiment said polypeptide shows a differential migration behavior.

Preferably, said two-dimensional polyacrylamide gel electrophoresis is in the first dimension an immobilized pH-gradient electrophoresis and in the second dimension a SDS polyacrylamide gel electrophoresis.

In order to isolate a polypeptide of the invention, the HL 60 cell line is cultured under conventional conditions. The cells are in the proliferative phase (not synchronous) and are at a cell concentration in the range of 0.3 to 1.0x10⁶ cells/ml, preferably at a cell concentration of about 0.5x10⁶cells/ml, staurosporine.

Stuarosporine is added to an appropriate final concentration, which is preferably in the range of 50 nM to 1mM, 1mM being preferred. The cells are cultured for an appropriate time, i.e. until a cellular condition related to apoptosis can be detected. Samples can be taken from the culture for monitoring the cell count and the cellular conditions. Cellular events which are markers for a cellular condition related to apoptosis are known in the art, for example condensation of nuclear material, collapse of the nucleus, continued integrity of cell organelles, or shrinkage of the cell itself (see e.g. K. G. Krul et al., (Spectrum Therapy Markets and Emerging Technologies, Decision Resources Inc., 1994, pp. 60-1 - 60-19; CM. Payne et al., Leuk-Lymphoma 19 (1-2) (1995), pp. 43-93). A cellular condition related to apoptosis can be obtained in HL 60 by continuing cultivation after addition of staurosporine for at least 3 hours, preferably for 3 to 18 hours, more preferably for 10 or 12 hours. After cultivation, the cells are collected and lysed. The lysate can be either analyzed directly or stored by immediately freezing at -80°C. The lysate can be analysed by 2-dimensional electrophoresis, being in one dimension, which preferably is the first dimension, an immobilized pH gradient electrophoresis (IPG) on IPG stripes which are commercially available. The gradient can for example be a non-linear pH gradient in the range of pH 3 to 10 or a linear pH gradient in the range of pH 4 to 7, the former being preferred. The other dimension, which preferably is the second dimension, can be SDS polyacrylamide gel electrophoresis, preferably on a slab gel, more preferably on a vertical slab gel, which for example is a gradient gel of 9-16%T / 2.6%C (T = total acrylamide concentration (w/v); C = percentage of crosslinker (PDA, piperazine diacrylyl) from total acrylamide), or a homogenous gel of 14% T / 2.6%C, the latter being preferred. The gel may lack a stacking gel.

Depending on the protein content of the cell lysate loaded onto the first dimension gel, the eventually obtained 2 dimensional gel can be used as an analytical gel suitable for subsequent differential image analysis (see below) or as a preparative gel suitable for subsequent amino acid sequence analysis (see below) or isolation of the polypeptide. The amount of lysate loaded onto the first dimension gel of an analytical gel having a size of 16cmx16cmx0.15cm contains preferably 0.2 to 0.5 mg protein, and of a preparative gel of the same dimensions preferably 0.5 to 3 mg protein, for example 1 mg.

For detection or identification of polypeptides the gels can be stained. In case of an analytical gel suitable in particular for subsequent differential image analysis for example silver staining, in particular silver staining according to B. Bjellqvist et al., Electrophoresis 14 (1993), pp. 1357-1365, can be performed; in case of a preparative gel for example silver staining, in particular silver staining according to A. Shevchenko et al., Anal. Chem. 86 (1996), pp.850-858, or Coomassie staining can be performed.

Polypeptides showing a differential expression pattern or differential migration behavior can be identified by comparison of a thus obtained analytical gel with another gel prepared as outlined above, however, using a cellular lysate of the non-induced HL 60 cell line. Such comparison can be for example performed by differential image analysis. The gels, comprising the cell lysate of the induced or non-induced cell line, respectively, can be scanned with an imaging densitometer. Differential display analysis can be performed by computer assisted image comparison of the gels. For calibration of a gel, in particular for determination of the pi value or molecular weight of a polypeptide according to the present invention, comparison with standard polypeptides, which may be run on a comparative gel, or comparison with known endogenous polypeptides, which may be used as landmark polypeptides in the same gel, can be performed.

A polypeptide, which has been identified to show a differential expression pattern or differential migration behavior on an analytical gel, can be isolated by convenient methods from the matrix of a corresponding preparative gel. For example, it may be possible to excise a gel slice containing the polypeptide of interest from the gel and to extract the polypeptide directly from the gel slice (see e.g. Hunkapillar et al., Methods in Enzymology 91 (1993), pp. 227-236).

In a particular preferred embodiment the polypeptides of the present invention are markers or surrogate markers for monitoring a cellular condition achievable by application of staurosporine, which cellular condition in particular is a condition related to apoptosis. Accordingly, in a yet further preferred embodiment said polypeptides are involved in induction or inhibition of apoptosis. According to the process of the present invention polypeptides can be identified upon their differential expression pattern or differential migration behavior and subsequently isolated^*. Accordingly, the present invention relates to a polypeptide obtainable by the process according to the present invention. In particular, the present invention relates to a polypeptide as claimed in claim 1.

The polypeptides of the present invention can be further analysed for their respective amino acid sequences according to conventional methods known to those skilled in the art. For example, a polypeptide can be blotted onto a suitable membrane, for example a PVDF membrane, and analysed by Edmam degradation (see e.g. G. Allen, in "Sequencing of Proteins and Peptides", T.S. Work et al., (eds.), Elsevier, Amsterdam, New York, (1981); R. Aebersold et al., Electrophoresis 11 (1990), pp. 517-527). Conveniently, such an analysis can be performed on an automated sequencer. In case the N-terminus of the polypeptide is blocked, the polypeptide may be cleaved, the cleavage products subsequently being sequenced by Edman degradation. Another possibility of amino acid sequence analysis can be MS/MS analysis of peptides, with for example a nano electrospray ion source (see e.g. M. Wilm et al., Nature 379 (1996), pp. 466-469). For analysis by mass spectrometry the polypeptide of interest can be used uncleaved or, preferably, cleaved. Such a fragmentation for example may be performed by enzymatic digestion, for example by tryptic digestion. A digestion for example may be performed as in-gel digestion by excising a polypeptide spot of interest from a preparative gel, if appropriate performing one or more washing and drying steps, and applying trypsin. After digestion, the polypeptide cleavage products can be extracted from the gel pieces and analyzed using nano electrospray ionization on an appropriate mass spectrometer. First, their molecular weight is determined, then the polypeptide cleavage products are submitted to MS/MS analysis in order to obtain sequence information. The spots can also be analyzed using the MALDI peptide map approach

Once the partial or complete amino acid sequence of a polypeptide of the present invention has been obtained, it is possible to check its identity by searching/comparing in an appropriate database, for example Swissprot or Genpept.

The polypeptides of the present invention are valuable tools for elucidating cellular effects caused by application of staurosporine and pathway analysis. Accordingly, the present invention further relates to the use of a polypeptide of the present invention as a marker or surrogate marker for monitoring a cellular condition achievable by application of staurosporine. Preferably said cellular condition is a condition related to apoptosis. If, for example, application of staurosporine induces the de-novo synthesis of a protein, such a protein may for example be detected by western blot analysis and immunodetection. If, for example, application of staurosporine induces a postranslational modification of a protein, such a modified protein may be analyzed by 2D-gel electrophoresis and/or western blot analysis (western blot analysis for example is described in D. Garfin et al., in "Protein blotting: methodology, research and diagnostic applications", Baldo Tovey (ed.), Karger, Basle, (1989), pp. 5-41).

Staurosporine, being known as an inductor of apoptosis, can have an inductive effect on a polypeptide according to the present invention, said polypeptide being an inductor of apoptosis. On the other hand, staurosporine can have a suppressive effect on a polypeptide of the present invention, said polypeptide being a suppressor of apoptosis. Therefore, a polypeptide of the present invention is suitable either for use as a drug in the treatment of a disease responsive to induction of a cellular condition achievable by application of staurosporine, or for use as a drug in the treatment of a disease responsive to inhibition of a cellular condition achievable by application of staurosporine, said cellular condition preferably being a condition related to apoptosis. Accordingly, a further object of the present application is a polypeptide of the present application for use as a drug.

A preferred embodiment of the present invention relates to a pharmaceutical composition for treatment of a disease responsive to induction of a cellular condition achievable by application of staurosporine to the cell, said composition comprising a polypeptide capable of inducing a cellular condition achievable by application of staurosporine to the cell. In a preferred embodiment the polypeptide capable of inducing a cellular condition achievable by application of staurosporine to the cell is obtainable by the process according to the present invention. A further embodiment relates to the use of a polypeptide of the invention in the preparation of a pharmaceutical for treatment of a disease responsive to induction of a cellular condition achievable by application of staurosporine to the cell, preferably wherein the cellular condition is a condition related to apoptosis.

A preferred embodiment of the present invention relates to a pharmaceutical composition for treatment of a disease responsive to inhibition of a cellular condition achievable by application of staurosporine to the cell, said composition comprising a polypeptide capable of inhibiting a cellular condition achievable by application of staurosporine to the cell. A further embodiment of the present invention relates to the use of a polypeptide capable of inhibiting a cellular condition achievable by application of staurosporine in the preparation of a pharmaceutical composition for treatment of a disease responsive to inhibition of a cellular condition achievable by application of staurosporine. In a particular preferred embodiment, said cellular condition is a condition related to apoptosis. In a further particularly preferred embodiment the polypeptide is obtainable by the process according to the present invention,

Said disease responsive to inhibition of a cellular condition achievable by application of staurosporine, said cellular condition preferably being a condition related to apoptosis, preferably is for example selected from the group consisting of (i) neurodegenerative diseases or neuropathies, in particular Parkinsons's disease, Alzheimer's disease, Huntington's disease, multiple sclerosis, (ii) immunodeficiencies, in particular AIDS or T-ceil depletion, (iii) geriatric diseases, and (iv) transplantation rejection, in particular chronic, long term rejection and rejection of xenotransplants. A disease responsive to induction of a cellular condition achievable by application of staurosporine, said cellular condition preferably being a condition related to apoptosis, preferably is for example selected from the group consisting of (i) hyperproliferative diseases, in particular cancer, particularly solid tumors and lymphomas, (ii) autoimmune diseases, in particular rheumatoid arthritis, and (iii) dermatological disorders, in particular psoriasis.

A polypeptide of the present invention, can also be used in the diagnosis of such a disease. Accordingly, the present invention relates to the use of a polypeptide of the present invention as a diagnostic marker or surrogate marker for a disease, said disease being selected from the group consisting of diseases responsive to induction of a cellular condition achievable by application of staurosporine, and diseases responsive to inhibition of a cellular condition achievable by application of staurosporine, wherein said cellular condition preferably being a condition related to apoptosis. An appropriate diagnostic assay can for example be a western blot assay or an ELISA, in classical or microchip based format (see e.g. M. Schena et al., Science 270 (1995), pp. 467-470).

A polypeptide of the present invention may also be used as a target for identifying one or more further compounds which induce or suppress the cellular effect of said polypeptide and thus are suitable for use as drugs themselves. Therefore, the present invention relates to the use of a polypeptide of the present invention as a drug discovery target for identification of a drug for treatment of a disease, said disease being selected from the group consisting of diseases responsive to induction of a cellular condition achievable by application of staurosporine, and diseases responsive to inhibition of a cellular condition achievable by application of staurosporine, wherein said cellular condition preferably is a condition related to apoptosis. An appropriate drug discovery assay can for example be a protein binding assay for screening for interacting compounds, preferably in microtiter plates, like, e.g., the yeast two-hybrid system according to M. Yang et al., Nucleic Acids Res. 23 (1995), pp. 1152-1157, a western blot assay, an ELISA or a scintillation proximity assay (Packard).

The amino acid sequence of a polypeptide according to the present invention, determined as described above, for example by mass spectrometry sequencing, if comprising about 5 amino acids or more, can be used to synthesize an oligonucleotide capable of specifically hybridizing with a nucleic acid coding for the polypeptide in question, for example a naturally occurring DNA, a RNA derived from said DNA, or a cDNA, which may be double- stranded or single-stranded. The oligonucleotide can for example be employed as a probe to screen a cDNA library for a clone coding for the polypeptide of interest. Hence, a further embodiment of the present invention relates to an oligonucleotide capable of specifically binding to a naturally occurring DNA or RNA, or a cDNA, coding for a polypeptide of the present invention. If appropriate, such an oligonucleotide can be provided with an appropriate label. An oligonucleotide according to the present invention for example has a length of 15 to 100 nucleotide or nucieoside building blocks. A nucleotide or nucleoside building block can be a naturally occurring nucleotide or nucleoside building block, such as a ribonucieotide, ribonucleoside, deoxyribonucleotide, or deoxyribonucleoside, or where appropriate, it can be a synthetically modified nucleotide or nucleoside, bearing, for example, a 2'-modification. The internucleosidic linkages of an oligonucleotide according to the present invention can for example be of the naturally occurring phosphodiester type. In the alternative, the linkages can be of the synthetic phosphothioate type or of another synthetic, modified linkage type. Synthetic, modified oligonucleotide building blocks and modified internucleosidic linkages are known in the art (see e.g. A. De Mesmaeker et al., Ace. Chem. Res. 28 (1995), pp. 366-374). An oligonucleotide according to the present invention can completely or in part comprise synthetic, modified building blocks or modified internucleosidic linkages, any remaining building blocks or internucleosidc linkages being of a naturally occurring type. Oligonucleotides bearing one or more of such synthetic, modified building blocks or linkages often have the advantageous property of increased nuclease resistance or increased hybridization affinity, rendering the resulting oligonucleotide extremely suitable for the intended use as a probe or primer, but also for use in antisense- technology.

The oligonucleotide according to the present invention can be used in a method for screening a cDNA library. When a clone is found it can be analyzed by sequencing. Accordingly, the present invention further relates to a DNA molecule encoding a polypeptide according to the present invention. The reading frame of the DNA molecule may be transferred into a suitable expression vector. This vector may be transfected into an appropriate host cell for recombinant protein production. Such methods are well known to those of skill. The recombinant protein can be isolated and used for structural or functional studies, or as a drug. A polypeptide according to the present invention, or a fragment thereof, can be used to immunize an animal for antibody production. The polypeptide or the fragment thereof can for example be prepared synthetically. Preferentially a hydrophilic fragment is selected because it is known in the art that a hydrophilic fragment will lie with a high probability at the surface of the corresponding polypeptide and very likely will serve as a good epitope, so that a thus produced antibody might have a high affinity for the polypeptide. Accordingly, the present invention further relates to an antibody directed to a polypeptide according to the present invention. Said antibody preferably is selected from the group consisting of a polyclonal antibody or a monoclonal antibody.

From an analysis of the localization of differential gene expression or modification of gene products (i.e. identification of the polypeptides of the present invention) we can comment as follows. Typically between 1000 and 2000 protein spots are detected in a silver stained 2D- map from whole cell lysates which is focused on a broad range non linear 3-10 or linear 3- 10 IPG-strip for 2DE and analysed with the Melanie II software. Using non linear broad range IEF 3-10 for the comparison of control cells vs induced cells 19 protein spots, namely 1 B to 19B, that are only present in lysates of STS-treated cells (Table 1) are observed Similarly, spots 1 A to 18 A are observed (data not shown). Most of these spots appeared 3 to 6 hours after addition of 0.5mM STS. From the STS-inducible spots, As is shown in Table 1 below, 19 spots (including landmarks L1 to L6) have been identified by the nano- electrospray/sequence tag approach alone. Two of those protein spots have later been submitted to the MALDI/peptide map approach in order to obtain a better sequence coverage. The 6 other spots have been submitted to MALDI/peptide map approach and 5 spots have thus been identified. The remaining spot, spot 16B needed the second stage of the combined approach for identification.

Table 1:

2D-gel-derived proteins identified

The accession numbers are all from the GenePept. database, except for the ones starting with the letter P which are from the SwissProt. database.

Three spots are isolated from the lysate of control cells, (17B, 18B and 19B) that are downregulated by more than two-fold upon addition of STS to the cells. Cofilin (spot 17B) is strongly downregulated in STS treated cells. From the apparent molecular weight (19kDa) the spot corresponds to the full length protein. Cofilin is a member of the small actin- binding proteins and it may play an essential role in promoting the extent of actin polymerization /depolymerisation (Lappalainen et al., (1997) Nature 388: 78-82). The actin associated activities of cofilin are regulated by pH, phosphorylation and phosphoinositides. The type of modification of cofilin (extent of phosphorylation) therefore might correlate with the morphology of the cell and thus be changed in apoptotic cells. Evidence has also been shown that dephosphorylation of cofilin plays an important role in activation of superoxide- generating enzyme in neutrophil-like HL60 cells (Suzuki, K. et al., (1995) J. of Biol. Chβm. 270 (33) 19551-19556). Nothing is known about the upstream mediators of cofilin modification. Since the STS-induced downregulation of the cofilin-spot could not be prevented by addition of ICE inhibitor the possible modification of cofilin may be an upstream-event from ICE activation or it may play an ICE-independent role. Ly-GDI (also named Rho-GDI-2 or D4-GDI) is identified in three different STS-induced spots (no.1A,1B, no.2A,2B and no.12A,3B). Ly-GDI is a hematopoietic cell specific homologue of rho-GDI, an inhibitor of Rho family GTPases (Lelias, J.-M et al (1993) Proc. Natl. Acad. Sci. USA 90: 1479 - 1483).

It has recently been shown that Ly-GDI is specifically cleaved by CPP32 during Fas- or STS-induced apoptosis at position Asp19 (Na, S., Chuang, et al., (1996) J. Biol. Chem. 271 (19): 11209 - 11213) and cleavage by ICE at position Asp55 resulting in truncated Ly- GDI that is unable regulate Rho GTPase in inflammatory leukocytes ( Danley-DE; et al., (1996) J-lmmυnol. 15; 157(2): 500-3.). The first 19 N-terminal amino acids for spot 1A,1B and 2A.2B of Ly-GDI were not detected in the nanoESI-MS measurements. In the STS- treated HL60 cells the truncation of Ly-GDI at position Asp19 was observed, since Edman sequencing of spot 1 blotted to a PVDF membrane revealed SβrLysLeuAsn or SKLN (SEQ. ID. NO. 3)- as N-terminal sequence. The apparent mass of full length Ly-GDI in SDS- PAGE has previously been shown to be 28.1 kDa, but we observed a mass of 22kDa for spot 1A.1 B (apparent pi: 6.4, apparent M.W. 22kDa) and 2 (apparent pi: 6.8, apparent M.W. 22kDa). Although we do not wish to be bound by our theory, compared to the theoretical pl=4.9 of Ly-GDI the basic shift of the truncated forms (pi 6.5) might have resulted from the loss of the N-terminal part that contains several acidic (Asp and Glu) residues.

The third GDI-spot (spot no. 12A, 3B, apparent pl:5.1 , apparent M.W. 5kDa)) corresponds to a small 5-6kDa fragment of the protein. This fragment includes the potential ICE- recognition site (position 55) in the non cleaved form. The observation we have made with the GDI-spots 1A.1B and 2A.2B strongly indicate that this protein is proteolytically cleaved during STS-induced apoptosis as has previously been described for Jurkat cells. Spots 4B and 19B have been identified as both being human adenylate kinase. In fact, the data match with the sequences of two isoforms of the human adenylate kinase (adenylate kinase 2; accession number : U39945; Mr. : 26477.88 Da; human adenylate kinase 2B; accession number : U54645; Mr. : 25614.72 Da), which differ only by their C-terminus. The sequence of the variant 2 has an extended C-terminus with the sequence CKDLVMFI (SEQ. I. NO. 14) instead of the C-terminal serine residue of the 2B variant. Adenylate kinase isoform 2 (ADK-2) was identified both in control cells (spot 19B) and in STS treated cells (spot 4B), but in STS treated cells we observed that (and increasing fraction of) the protein undergoes a pi shift from about pi 7.9 to pi 7.6. The ratio of low pi ADK-2 (Spot4B) to high pi ADK-2 (Spot 19B) increased with time of STS exposure. By nanoESI-MS the N- terminal peptide for both ADK spots is identified. The more basic form of ADK (pi 7.9) that was downregulated in the STS treated cell culture begins with alanine, which is the correct N-terminus as expected from the gene sequence. The more acidic spot which was upregulated when STS is added to the cells starts with a serine at the N-terminus. The serine corresponds to amino acid no3 in the ADK reading frame. The calculation (Bjellqvist, B. et al., (1993) Electrophoresis, 14, 1023-1031.) of the pi's for the two variants of ADK shows a very comparable difference as observed for the two corresponding spots in the 2D- gels. Therefore the STS induced apoptosis of HL60 cells seems to be associated with a truncation of ADK-2 by the first two amino acids. Since the dipeptide alanine/proline at the N-terminus of proteins is a typical substrate for DPPIV we quantified the DPPIV activity in HL60 cell lysate from controls vs STS treated. Dipeptidyl peptidase IV (DPPIV) is a cell surface type serine protease well known as CD26 and expressed on a variety of different cell types, e.g. epithelial cells of the intestine, prostatic gland and kidney proximal tubules. CD26/DPPIV is more prominent for its increased expression on activated T cells but the physiological substrate of CD26/DPPIV is still not known. CD26/DPPIV cleaves dipeptides from the N-terminus of peptides/proteins, while the second amino acid has to be a proiine there is a strong preference for glycine or alanine in the first position (Ikehara, Y. et. al., (1994) Methods in Enzymology, 244: 215-227). We measured DPPIV activity using a fluorogenic substrate ala/proCMAC. Whereas in the supernatant of PBS-washed HL60 cells of both control cells and cells treated with 0.3uM STS for 9 hours the DPPIV activity was at a similar level we measured a higher DPPIV activity in extracts of STS treated cells. Taking into consideration that the protein extract is prepared from STS-treated cells representing a cell-mixture being at different phases of the cell cycle or at different progression steps of apoptosis the observed difference in DPPIV activity is more significant. The DPPIV activity from the cell lysates was inhibited by DiprotinA in a similar manner as isolated liver DPPIV was inhibited. The enhanced DPPIV activity in apoptotic HL60 cells could either be attributed to the induction of so far unknown but similar soluble intracellular protease or to an intracellular accumulation of CD26/DPPIV due to an altered protein processing and targetting in apoptotic cells. It has previously been shown that DPPIV appeared upregulated in thymocytes that undergo apoptosis independent from viral infection (Ruiz, Ph. et al. (1996) Cytometry 23, 322-329). The spots nos. 7B, 8B and 9B are derived from N-terminal fragments of laminA. laminBI and B2. Lamin is known to be a substrate of proteases during apoptosis. It has previously been observed that in a later stage of apoptosis laminA is the substrate of an ICE-like protease that generates a 45kDa fragment (Larsson, N. et al. (1995) J. of Biol. Chem. 270(23), 14175-14183)

In thymocytes laminB degradation is an event in apoptosis followed by chromatin condensation and breakdown of the nuclear envelope (Beretta, L. et al. (1995) Eur. J. Biochem. 227, 388-395).

The STS-induced spot no. 11 B corresponds to a fragment of a-tubulin. Spot no5B which appears only in 2DE from induced cells corresponds to a 15kDa fragment of nucleolin which is known with various molecular masses, ranging from 76kDa to 105kDa (Belmont L et al., (1996) Cell 84, 623-631). It is a nuclear protein with an RNA and a DNA binding domain. The protein is associated with nuclear chromatin and preribosomal particles in growing cells. It is thought to be involved in r-RNA transcription ribosomal assembly and translocation of ribosomal proteins. It was also observed that nucleolin has self cleaving activity depending on the proliferative state of the cell and other work indicates that nucleolin is cleaved into several peptides by endogenous proteases. In target cell apoptosis mediated by cytotoxic T lymphocytes, nucleolin has been observed as a substrate of granzyme A (Smyth, M., et al., (1994) Clin. Exp. Pharmacol. Physiol. 21 , 67-70). In our case nucleolin which plays some key roles in the nucleus may also be a target for specific degradation in the apoptotic process.

We identified a fragment of arachidonate 15-lipoxygenase (15-LOX=spot10B) that only appears after STS treatment. 15-LOX generates metabolites from arachidonic acid which are involved in the transduction of growth related signals and regulation of cell proliferation. It was recently suggested that 12-LOX and probably also 15-LOX are involved in the regulation of cell survival (Tang, D. et al. (1996) Proc. Natl. Acad. Sci. USA 93, 5241-5246). W256 cells treated with antisense oligonucleotides directed to 12-LOX underwent time- and dose dependent apoptosis. Cells treated with 5-LOX inhibitors commit apoptosis (Anderson, K. et al., (1995) Leυk. Res. 19(11), 789-801). Likewise the specific inactivation of LOX by proteolytic cleavage may be a possible in vivo mechanism during apoptosis. One of the peptides identified by nanoESI-MS has an N-terminus that could not be generated by tryptic cleavage. The cleavage of AIKD SL resembles an ICE-like recognition site. An array of three spots (no.15B) with the same apparent molecular weight of 115kDa and a pi of approximately 4.5 is observed in lysates of STS treated cells only. These spots correspond to non-muscular myosin heavy chain. It is not known if the appearance of the spots in STS treated cells is due to a truncation or if the protein is differentially phosphorylated. MHC phosphorylation is observed in correlation with cytoskeletal reorganization and it may play a role in the regulation of myosin filament assembly and cellular localization (Egelhoff, T. T., et al. (1993) Ce//75, 363-371). One of several known MHC kinases (MHC-PKC) is homologous to PKC (Ravid, S., et al. (1992) Proc. Natl. Acad. Sci. USA 89, 5877-5881) and might therefore also be inhibited by STS. The identification of spot no. 6B revealed an isoform of stathmin (it has some other names: p17, p18, Op18, p19, 19K, prosolin, metablastin). The protein contains at least four phosphorylation sites (Lazebnik, Y.A., et al. (1995) Proc. Natl. Acad. Sci. USA 92: 9042 - 9046) and its state of phosphorylation and expression level is regulated by a variety of cellular effectors (Neamati, N., et al. (1995) J. Immunol. 154(8): 3788 - 3795). The phosphorylation of the protein fluctuates with the cell cycle state and it's expression is elevated in leukemic cells. Taken altogether, stathmin represents a phosphorylation switchpoint for a variety of secondary messenger pathways. In our case the STS-induced stathmin-spot may correspond to the full length protein. In the tryptic peptides detected by nanoESI-MS we could see phosphorylation at neither of the possible sites Ser16, Ser25 and Ser38. Also the apparent pl=5.6 of spotθ is equal to the theoretical pi of stathmin in the completely dephosphorylated form. It is very likely that in the cellular extracts stathmin is present in various, differently phosphorylated forms simultaneously and the faint spot 6B represents only a minor component that is favored in apoptotic cells. Evidence has been shown in earlier findings that stathmin is a PKC substrate (Kharrat, A., et al. (1991 ) Biochemistry 30, 10329-10336) and that inhibitors of PKC block the phosphorylation of stathmin (Bugler, H. et al. (1987) J. Biol. Chem. 262, 10922-10925). Apart from the above discussed changes, the 2D-PAGE protein pattern of control vs STS- treated cells remained very similar even 15 hours after addition of 0.5mM STS which is remarkable since at this time more than 90% of the cells were in a progressed apoptotic state as judged by Pl-staining.

From the apparent molecular weights that are significantly reduced compared to the theoretical mass of most of the STS-induced and identified spots (table 1 ), we conclude that these protein spots correspond to truncated forms of the proteins (e.g. Ly-GDI, lamin- variants, a-tubulin, 15-lipoxygenase, nucleolin and splicing factor SF3a120). The accumulation of these protein fragments may be a consequence of increased proteolytic activity in apoptotic cells.

Furthermore, the present invention in particular relates to the embodiments as described in the following examples. The examples are not intended to restrict the invention as outlined above.

Examples:

A) Materials:

40% acrylamide solution, piperazine diacrylamide (PDA), N, N, N\ N'-tetramethyl- ethylenediamide (TEMED), Triton X-100, sodium dodecylsulfate (SDS), are all purchased from BioRad and of "electrophoresis purity reagent" grade. Tris(hydroxymethyl)- aminomethan) (= Tris) (p.a.), Glycine (p.a.), ammonium vanadate (p.a.), methanol (p.a.), silver nitrate (p.a.), ammonium bicarbonate (p.a.), CaCl2 (p.a.), acetonitrile (gradient grade) and Bromophenol blue are all purchased from Merck. Urea (MicroSelect), 4-(2- Hydroxyethyl)-piperazine-1-ethane-sulfonic acid (HEPES, BioChemika), Dithiotreitol (DTT, BioChemika), sodium thiosulfate (purum p.a.), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS, MicroSelect), acetic acid (p.a.), formaldehyde (puriss, p.a.), and formic acid (p.a.) are all purchased from Fluka. Pharmalyte® and Immobiline® are purchased from Pharmacia Biotech. The staurosporine used is the Ciba-Geigy Product CGP39360, corresponding to the staurosporine which is commercially available from Calbiochem, La Jolla, CA, USA, Cat.-No. 569397-Q, which is stored at 10mM in DMSO.

Samples submitted for nano-Electrospray(ES)-analysis are prepared in prewashed original Eppendorf tubes. The tubes are washed in detergent, 2M nitric acid, then three times each in ultra pure water, acetone, methanol and methylenechloride.

B) Experimental part: Example 1 : Induction of apoptosis with staurosporine

The human HL60 promyelocytic leukemia cell line, which has been deposited with the Deutsche Sammlung von Mikrooorganismen und Zellkulturen (DSM), Braunschweig, FRG, on April 02, 1997, is propagated at 37°C (5% CO2) in RPMI1640 medium (Gibco BRL) supplemented with 5% ( v) fetal calf serum (Gibco BRL). Staurosporine, which is prepared in DMSO (dimethylsulfoxide) at 2000x concentration before addition, is added at a cell density of about 0.5 x 10⁶ cells/ml to a final concentration of 1mM. The cells are preferably induced 4 days after the last passage. After addition of staurosporine, cultivation is continued for 12 hours. Progress of apoptosis is measured by propidium iodine binding according to known technology when the cells are harvested, e.g. for subsequent 2D- analysis.

Example 2 Induction of apoptosis with staurosporine

Example 1 is repeated except that the cells are incubated for 10 hours rather than 12 hours and the staurosporine is prepared in DMSO at 500-1 OOOx of the final concentration in the culture.

Example 3: Preparation of cell lysates:

After cultivation the HL60 cell suspension is transferred into a tube and the cells are centrifuged at 1000 rpm during 5 minutes. The supernatant is discarded and the cells are resuspended in wash buffer (HEPES (25mM) pH 7.4, NaCI (150mM), NH4VO3 (0.2mM)) at 4°C. After centrifugation and removal of buffer the cell pellets are immediately dissolved in lysis buffer (urea (8 M), Triton X-100 (4% w/v), Pharmalyte® pH3-10 (2% w v),Tris (40 mM), DTT (130 mM) NH4VO3 (1mM) and 1 tablet of protease inhibitor cocktail (Complete™, Boehringer Mannheim) per 50ml buffer and a trace of Bromophenol Blue) at room temperature.2ml lysis buffer is added per 10⁸ cell equivalents. Solubiiization is performed by 5-10 strokes in a Potter-Elvehjem homogenizer. The lysates are subsequently centrifuged at 20000xg at 15°C for 20min to remove insoluble particles. The protein content of the lysates is determined using the Bradford protein assay from BioRad. If appropriate, the HL60 lysate is finally diluted in lysis buffer (see above), and is either immediately loaded onto IPG gel strips (see below) or immediately frozen at -80°C.

Example 4: 2-Dimensional gel electrophoresis a^ First dimension (separation of polypeptides according to their charge) (i) Immobilized pH Gradient (IPG) gel strips preparation:

Immobilized pH gradient (IPG) electrophoresis is used for the first dimension. IPG-strips with a non-linear pH gradient from 3-10 which are commercially available from Pharmacia Biotechnology, are used. The strips are 3 mm wide and 180 mm long. Hydration of the IPG strips is performed overnight in the Pharmacia reswelling cassette with 25 ml of a solution containing urea (8 M), Triton X-100(2% w v), DTT (10 mM), Pharmalyte® pH 3.5-10 (2% v/v) and a trace of Bromophenol Blue.

If desired, IPG-strips with narrower pH ranges can be prepared essentially as described in

(Westeremier, R., Electrophoresis in Practice, VCH, Weinheim (1993), pp. 197-207).

(ii) Running conditions:

IPG-strips, which have been rehydrated as described above are used for isoelectric focusing on the Multiphor II System (Pharmacia) according to standard protocols (Instruction manual for Immobiline DryStrip Kit, Pharmacia Biotech, Edition AB, 18-1038-63 (1994)).

Samples are applied in sample cups from Pharmacia for volumes up to 120ml, and for larger volumes (1 ml) home built sample cups (see below) are used. The 1 ml-sample cup is obtained by cutting a disposable polystyrene cuvette (1ml) at 2.3cm from its bottom. The upper part is then sealed to the mounting support of a 120ml sample cup (the 120ml cup has to be cut off in advance). IPG strips have to be 8mm wide at the sample application site if the large sample cups are employed.

The total protein amount loaded on is 0.2mg in 0.03ml solution per analytical gel, and 1mg in 0.12ml solution per preparative gel.

The voltage is set to 500V for 4 hours and then linearly increased from 500V to 3500V during 2 hours, followed by 24 additional hours at 3500 V. A total volthourproduct of 85-90 kVh or more is applied in a two days run.

(iii) IPG gel strips equilibration:

After the first dimension run the strips are equilibrated in order to resolubilize the proteins and to reduce -S-S- bonds. The IPG-strips are equilibrated in Tris-HCI (50 mM) pH 6.8, urea (6 M), glycerol (30%v/v; Rotipuran® from Roth, Karlsruhe), SDS (4% w/v) and DTT (100mM) for 10 min. -SH groups are subsequently blocked with a solution containing Tris- HCI (50 mM) pH 6.8, urea (6 M), glycerol (30% v/v), SDS (2% w/v), iodoacetamide (300mM, Sigma) for 10 min.

b) Second dimension (sodium dodecyl sulfate polyacrylamide-electrophoresis, separation of polypeptides according to their molecular weight) (i) SDS polyacrylamide gel:

In the second dimension a vertical homogenous (14% T/ 2.6% C) slab gel (dimension: 160 x 160 x 1.5 mm) with the Laemmli-SDS-discontinuous system (buffer: 0.375M Tris, pH 8.8) is used. The crosslinker is piperazine diacrylyl (PDA). 5mM sodium thiosulfate is added if analytical gels are prepared. 0.05% ammoniumpersulfate and 0.05% TEMED are added to initiate polymerization. The gels are cast at 20°C until 0.7 cm from the top of the plates and no stacking gel is used.

(ii) IPG gel strip transfer:

After the equilibration, the IPG gel strips are cut to size (160mm). The second dimension gel is over layered with a solution containing agarose (0.5% w/v; Type II, Sigma) and Tris- glycine-SDS (25 mM-198 mM-0.1% w/v) pH 8.3, heated to about 70°C and the IPG gel strips are immediately loaded through it.

(iii) Running conditions:

The gel is run in running buffer (Tris (25mM), glycine (198mM), SDS (0.1% w/v), pH 8.3) at 19°C for 3 to 4 hours. The applied current is 40 mA/gel (constant), the applied voltage is 100 to 400 V.

(iv) 2D-gel staining:

For analytical gels and subsequent differential image analysis the silver staining protocol described in Bjellqvist, B. et al., Electrophoresis 14 (1993), pp. 1357-1365, is used. In the alternative, in order to detect polypeptides of interest which cannot be stained by the silver stain procedure, analytical gels for protein identification by mass spectrometry are

Coomassie stained using the BM Fast Stain (Boehringer Mannheim).

For subsequent Edman sequencing or amino acid composition analysis the proteins are electrotransfered from the gel onto PVDF-membranes (Millipore). The transfer is performed in the Trans-Blot cell (BioRad) at 100V for 3hours in a buffer of CAPS (10mM) pH 11 , 10% methanol. Protein spots on PVDF-membranes are visualized by Coomassie stain as follows: incubating 15 sec. in 100% methanol, incubating 1 min. in a solution of 0.1% Coomassie Blue R-250 in 40% methanol/1% acetic acid, destaining in 50% methnol, rinsing in distilled H₂O.

Preparative gels prepared for protein identification by nanoESI-MS are silver stained with a modified procedure as follows. After electrophoresis the gels are fixed in 50% methanol/5% acetic acid for 20min. The next steps are: 50% methanol (10min), 100% water (10min), 0.02% sodium thiosulfate (1min), rinsed twice (1min) with water, 0.1% silver nitrate (20min at 4°C), rinsed twice with water and developed in 0.04% (35%) formaldyde in 2% sodium carbonate. The staining is stopped with 5% acetic acid. Alternatively the gels are Coomassie stained using the BM Fast Stain (Boehringer Mannheim).

Preparative gels for protein isolation are Coomassie stained using the BM Fast Stain (Boehringer Mannheim).

Example 5:

Example 4 is repeated except that in the immobilized pH Gradient (IPG) gel strips preparation used in the first dimension, IPG-strips with a non-linear pH gradient from 3-10 or a linear gradient from pH 4-10 are used and CHAPS (2% w/v) is used instead of Triton X- 100 . Also the running conditions of the first dimension were a total volthourproduct of 80-90 kvh for analytical and up to 120kvh for preparative gels. The temperature was 15C during the entire run. In the second dimension a vertical homogenous (14% T / 2.6% C) or 9-16% T/2.6%C slab gel (dimension: 160 x 160 x 1.5 mm) with the Laemmli-SDS-discontinuous system (buffer: 0.375M Tris, pH 8.8) is used.

Example 6: Differential display analysis

Analytical gels are scanned at 450dpi resolution with a GS-700 imaging densitometer (BioRad). Gel image comparisons for differential display analysis and protein spot quantification are performed using the Melanie II software (BioRad), which is installed on a Power Macintosh 95000/150 or UNIX workstation. Analysis is performed by comparison of an analytical gel containing polypeptides from a cell lysate of the induced HL 60 cell line with an analytical gel containing polypeptides from a cell lysate of the non-induced HL 60 cell line. Several total cell lysates were prepared from independently cultured and induced HL60-cells and at least three comparisons were performed to localize specific changes in the spot pattern.

For determination of the respective pi value and molecular weight of a polypeptide of interest the following standard polypeptides are used:

for the first dimension (all polypeptides are purchased from BioRad): soybean trypsin inhibitor: pi 4.5/21.5kDa;bovine Lactoglobulin: pi 4.8/18.2kDa;bovine serum albumin: pi 5.4-5.6/66kDa;bovine carbonic anhydrase: pi 6.0/31 kDa; equine myoglobin: pi

7.0/17.5kDa; and rabbit muscle GAPDH: pi 8.3-8.5/36kDa; for the second dimension (this is the molecular weight standard "broad range" of BioRad): rabbit muscle myosin: 200kDa;E.coli galactosidase: 116kDa;rabbit muscle PhosphorylaseB:

97.4kDa;bovine serum albumin: 66kDa;Hen egg white ovalbumin: 45kDa;bovine carbonic anhydrase: 31 kDa;Soybean trypsin inhibitor: 21.5kDa;Hen egg white lysozyme: 14.5kDa; andaprotinin bovine pancreas: 6.5kDa.

The standard proteins are mixed with the cell lysate to be analyzed and run on a 2D-gel as described above. The amount of standard polypeptides used is about 0.2 mg per polypeptide. By comparison of such a gel with a gel to be analyzed, bearing the cell lysate without the standard polypeptides, and, if appropriate, by further comparison with a corresponding 2D gel bearing only the standard polypeptides without cell lysate, the respective apparent pi value and apparent molecular weight of a polypeptide of interest is determined.

Upon differential display analysis performed as described using the gels polypeptides numbers 1A to 18A and 1B to 19B (referred to above) showing a differential expression pattern or a differential migration behavior are identified. It will be seen from the results below that the individual spots of the following pairs are the same since they have the same apparent pi and apparent molecular weight: 1A, 1B; 2A.2B; 10A.6B; and 12A.3B. Furthermore, it is visually observed that the spots of the following groups a, b, c, d, e, f, g, f and h, have very close apparent pi and apparent molecular weights and be the same, the differences probably resulting from the differences in experimental conditions: (a) 3A and 4B; (b) 4A.17A.18A and 15B; (c) 11 A and 5B; (d) 7A and 7B; (e) 8A and 10B; (f) 9A and 11 B; (g) 13A and 8B; and (h) 16A and 13B. Example 7: Amino acid sequence determination

Two different approaches are chosen for protein identification, i.e. Edman degradation or peptide analysis by mass spectrometry.

a) Edman degradation

Proteins blotted on PVDF-membranes are analyzed by Edman degradation on a Hewlett Packard G 1000A sequencer, (chemistry ver. 3.0).

b) Peptide analysis by mass spectrometry (i) In gel digestion:

The protein spot of interest are excised from a preparative 2D-ges and prepared for in-gel digestion in a prewashed Eppendorf tubes. After the respective gel pieces are excised, they are dehydrated in acetonitrile and subsequently dried in a Speed Vac concentrator. They are afterwards rehydrated in 50mM DTT, 50mM ammonium bicarbonate in a volume sufficient to cover the gel pieces and incubated at 56°C After 1 hour the DTT solution is replaced by 200mM iodoacetamide (Sigma) in 50mM ammonium bicarbonate and incubated at room temperature for 45min in the dark. The gel pieces are then washed in 50-100ml 20mM ammonium bicarbonate, shrunk by addition of acetonitrile, reswelled in 20mM ammonium-bicarbonate, dehydrated again in acetonitrile and completely dried in a speed Vac. The dried gel pieces are swollen in digestion buffer containing 20mM ammonium bicarbonate, 5mM CaCi2 and 12.5ng/ml of trypsin (sequencing grade, Boehringer Mannheim) in an ice cold bath for 45min. The supernatant is then removed and replaced with 5-10ml of the same buffer but without trypsin to keep the pieces wet during the digestion over night at 37°C

After digestion 20mM ammonium bicarbonate was added to cover the gel piece and after vortexing several times the buffer containing peptides was transferred to a clean Eppendorf tube. Further extraction procedures to gain more peptides from the gel were omitted because these steps very often introduced polymer contaminants. The peptide containing supernatant was then dried in a Speed Vac concentrator.

(ii) Concentration gels:

If the protein quantity obtained by extraction of t a spot is not high enough for its identification by mass spectrometric analysis, identical spots from several gels are pooled and the protein is concentrated on a funnel shaped gel. Such a gel is prepared as described in G. Lombrad-Platet et al., Biotechniques 15 (4) (1993), pp. 668-670, 672. From one gel two spacers(dimension 1.5x 5x83mm) are cut at an angle at one edge to produce a large funnel shaped slot and a small separating gel only 15mm high. The MiniProtean System is used to cast the funnel shaped polyacrylamide gels. The collected protein spots are washed in distilled water and equilibrated for 15 min (protein spots isolated from following procedure according to Examples 1, 3 and 4 above) or a minimum of 12 hours (protein spots isolated from following procedure according to Examples 2, 3 and 5 above)in running buffer and loaded into the funnel well. Electrophoresis is started at 5mA per gel and terminated when the Coomassie dye-front has reached 2-3mm down from the top of the separating gel. The protein band is visualized using the BM Fast Stain (Boehringer Mannheim). After cutting out the protein band is destained by incubating in 50% (v v) acetonitrile / 50mM NH HCO₃ at 30C for 2 - 3 hours. The destained gel piece is subsequently prepared for in-gel digestion as described above.

(iii) NanoES mass spectrometry (see Wilm et al (1996) Nature; 379, 466-469):

Needles for electrospraying are obtained from the Protein Analysis Company (Odense, Denmark). A dried protein digest, obtained as described above, is redissolved in 5% formic acid, concentrated and desalted on a capillary similar to the spraying capillary. This capillary is packed with 100nl of POROS R2 sorbent (Perseptive Biosystems, Framingham, MA) and equilibrated with 5% formic acid. After loading the dissolved peptide digest, the capillary is washed with 20ml of 5% formic acid / 5% methanol. The sample is eluted into the spraying capillary in two times 1ml of 5% formic acid/50% methanol. The spraying process is started by applying a voltage difference between the needle tip and the orifice (1.5 mm distance) of the mass spectrometer (API III, PE-Sciex, Toronto, Canada). A full mass spectrum is acquired over a wide mass range (m/z 350-1500) and subsequently parent ions of interest are mass-selected and fragmented by collision induced dissociation with argon (collision energy 96 eV; collision gas thickness 300x10¹⁴ molecules/cm²). (NanoEs is performed on an API III PE Sciex mass spectrometer, Toronto, Canada). Q-_j scans are performed with 0.1 Da mass resolution and mass steps. For operation in the MS/MS mode, Q-j is set to transmit approximately a mass window of 2Da.Since there is usually 1 h of measurement time per peptide mixture available, collision energy is tuned individually for each peptide to obtain maximum sequence information. The mass maps are then rapidly compared with the mass map obtained for a blank sample (piece of gel which does not contain proteins, but which is treated similarly as a spot containing proteins), and the molecular ions of interest are directly submitted to MS/MS analysis. Resolution is set so that fragment masses can be assigned to better than 0.5Da.

The mass-selected parent ions are fragmented by collision with argon. ("Since the 1 μL of solution allows to spray for at least 20 min., the collision energy in the MS/MS experiment is tuned individually for each peptide to obtain the best possible MS/MS spectra". From the MS/MS spectrum a short sequence of three or more amino acid residues can in general be deciphered. This short stretch (sequence tag) together with its position in the peptide as measured by MS/MS and additional information (see below) are sufficient to identify the peptide in the large sequence databases (Swissprot, Genpept.) by using the program Peptide Search ver2.9.2b1 ( see M. Mann, in: Microcharacterisation of Proteins, Kellner, R., Lottspeich, F. and Meyer, H.E. (eds.), VCH, Weinheim (1994), 223 - 245) running on a Macintosh computer. .

Finding a match is the first step of the procedure. As a second step, all expected fragment ion masses are calculated to verify the found sequence. If a number of possible sequences have been retrieved by the search, in general a close inspection of the MS/MS spectrum quickly reveals the correct match. Information about the source organism of the matching peptide can also be used. Furthermore, in general tandem spectra of several different peptides present in the cleavage mixture are generated. If those also provide the same hits with the above described procedure, the identification can be considered as established (M. Mann et al., Error-Tolerant Identification of Peptides in Sequence Databases by Peptide Sequence Tags. Anal. Chem. 1994; 66: 4390-4399).

1. Analysis of spot 2A.2B (a polypeptide having on a 2D-oel an apparent pi value of 6.8 and an apparent molecular weight of 22 kDa) using NanoES mass spectrometry

in the following, a detailed analysis of the data obtained for a peptide present in tryptic digest of spot 2A.2B is presented. Spot number 2 is identified At first a mass map of the tryptic digest is generated by nano-electrospay MS (Fig. 1 , upper trace). After assigning background signals by comparison with identically treated blank spots, the molecular ions of interest, in this case m/z 656.4, were mass selected and fragmented by collision-induced dissociation (CID) with argon. Under low-energy collision conditions fragmentation of peptides occurs mainly at the amide bound of the peptide backbone generating a ladder of sequence ions. Charge retention on the carboxy-terminal end produces so-called y-ions, whereas fragments with charge retention on the amino-terminal side form b-ions (Fig. 2) ( Roepstorff, P et al. (1984) Biomed. Mass Spectrom. 11, 601-603; Biemann, K. (1988) Biomed. Environ. Mass Spectrom. 6, 99-111 ).

Generally a short sequence stretch of 3 or 4 amino acids can be assigned. In Fig. 1 (lower trace) such a short stretch, GDGI/L (SEQ. ID. NO. 1), is formed by the fragment ions m/z 755.4, 812.2, 927.4, 984.4 and 1097.6 (L and I as well as K and Q can not be differentiated by the applied technique). At that stage the direction of the sequence remains open and might be as well LJIGDG (SEQ. ID. NO. 2). Usually C-terminal series are more prominent since trypsin cleaves after basic arginine or lysine residues, which favor C-terminal charge retention. Therefore the sequence was assumed to be LJIGDG (SEQ. ID. NO. 2) (from N- to C-terminus). The start mass of the fragment series (755.4) and its end (1097.6) allow together with the known mass of the peptide (1310.8 Da) the calculation of the mass differences to the N- and C-terminus (ml and m3), which form together with the sequence stretch the sequence tag (ml) LJIGDG (m3). This tag is used to search the sequence data base GenePept (Peptide Search program from M. Mann, EMBL Heidelberg applied to GenPept database, Mann, M., In Microcharacterization of Proteins; (1994) Kellner, R., Lottspeich, F., Meyer, H. E., Eds.; VCH: Weinheim, 223-245.) together with some restriction, e.g. regular tryptic cleavage, 0.1% mass accuracy, parent protein molecular mass up to 300 kDa. Two hits were obtained as a result of the search, which represent two database entries of the same protein, Ly-GDP dissociation inhibitor (Ly-GDI) (see Fig. 3). For verification, masses of fragment ions are predicted for the retrieved peptide sequence and compared with those present in the spectrum (cf. Table 2, see below). In this way 9 of 13 amino acids were assigned (Fig. 3). Without unassigned peaks of high intensity, this result represents already an unambiguous protein assignment. Nine additional peptides have been analyzed in a similar way. They all matched the sequence of Ly-GDI and covered 57 % of the amino acid residues of the protein. Table 2:

Calculated fragment ions of sequence TLLGDGPWTDPK(SEQ. ID. NO. 3). The fragment ions present in the MS/MS spectrum are marked in bold.

Experimental Mr : 1310.8 Da Calculated monoisotopic Mr : 1310.72 Da Calculated isotopically averaged Mr : 1311.51 Da

2. Analysis of spot 3A using NanoES mass spectrometry

The procedure used for the analysis of spot 2A.2B as outlined above is applied to spot 3A.

Searching of the Genpept. data base, performed for nine peptides present in that digest permitted to identify two proteins of human origin which amino acid sequences match with the MS/MS-data obtained (human adenylate kinase 2; accession number : U39945; Mr. : 26477.88 Da ; human adenylate kinase 2B; accession number : U54645; Mr. : 25614.72 Da). These two proteins have very similar amino acid sequences, they are variants. For each analyzed peptide the MS/MS data and the additional information used to identify the protein(s) by the program PeptideSearch are summarized hereafter.

MS/MS data and information used to perform the search for signal at m/z = 666.5 :

Peptide tag used to identify the protein: (501.2)EIIP (953.4) (SEQ. ID. NO. 4) data bank : GenPept. release 97.

M_r (monoisotopic) = 1996.5 / mass accuracy : 0.1%

Tryptic digest (N- and C -terminal specificity) l=L and Q=K, cysteine modified to carboxamidomethylcysteine y-type fragment ion series / Mr. of the searched protein < 100 kDa

MS/MS data and information used to perform the search for signal at m/z = 568.8 :

Peptide tag used to identify the protein: (476.2)DII(817.6) data bank : GenPept. release 97.

M_r (monoisotopic) = 1135.6 / mass accuracy : 0.1%

MS/MS data and information used to perform the search for signal at m z = 715.5 :

Peptide tag used to identify the protein: (684.4)TIDDQ(1256.8) (SEQ. ID. NO. 5) data bank : GenPept. release 97.

M_r (monoisotopic) = 2858.0 / mass accuracy : 0.1%

MS/MS data and information used to perform the search for signal at m/z = 505.4 :

Peptide tag used to identify the protein: (570.2) YEPEAA(1230.8) (SEQ. ID. NO. 6) data bank : GenPept. release 97.

M_r (monoisotopic) = 1513.2 / mass accuracy : 0.1 %

Tryptic digest (C -terminal specificity only) l=L and Q=K, cysteine modified to carboxamidomethylcysteine y-type fragment ion series / Mr. of the searched protein < 100 kDa

MS/MS data and information used to perform the search for signal at m/z = 530.8 :

Peptide tag used to identify the protein: (629.2)KGAGP(1039.4) (SEQ. ID. NO.7) data bank : GenPept. release 97.

M_r (monoisotopic) = 1589.4 / mass accuracy : 0.1%

MS/MS data and information used to perform the search for signal at m/z = 603.9 :

Peptide tag used to identify the protein: (941.8)IDDK(1412.6) (SEQ. ID. NO. 8) data bank : GenPept. release 97.

M_r (monoisotopic) = 3014.5 / mass accuracy : 0.1%

MS/MS data and information used to perform the search for signal at m/z = 698.1 :

Peptide tag used to identify the protein: (476.2)DII(818.0) data bank : GenPept. release 97.

M_r (monoisotopic) = 2091.3 / mass accuracy : 0.1%

MS/MS data and information used to perform the search for signal at m z = 909.6 :

Peptide tag used to identify the protein: (813.6)ISFEI(1403.6) (SEQ. !D. NO. 9) data bank : GenPept. release 97.

M_r (monoisotopic) = 1817.2 / mass accuracy : 0.1%

Tryptic digest (N- and C -terminal specificity) l=L and Q=K, cysteine modified to carboxamidomethylcysteine y-type fragment ion series / Mr. of the searched protein < 100 kDa MS/MS data and information used to perform the search for signal at m/z = 572.7 :

Peptide tag used to identify the protein: (175.0)IIP(498.2) data bank : GenPept. release 97.

M_r (monoisotopic) = 2858.5 / mass accuracy : 0.1%

In order to distinguish whether spot 3A relates to human adenylate kinase 2 or 2B, the molecular mass of the intact polypeptide contained in spot 3 is determined by mass spectrometry and compared with the known molecular weight of human adenylate kinase 2 or 2B, respectively.

(iv) Analysis of spot 12B bv MALDI/peptide map approach

The principles of the approach (Mann, M., In Microcharacterization of Proteins; (1994) Kellner, R., Lottspeich, F., Meyer, H. E., Eds.; VCH: Weinheim, 223-245). and the different steps involved are exemplified with the data from the analysis of spot 12B.

Sample preparation a-cyano-4-hydroxy-cinnamic acid/nitrocellulose matrices are prepared by the fast evaporation technique according to Jensen et al (1996 Rapid Commun. Mass Spectrom. 10, 1371-1378.). A saturated solution of a-cyano-4-hydroxy-cinnamic acid (Sigma, Buchs, Switzerland) in acetone was mixed in a 4:1 ratio with a solution of nitrocellulose in isopropanol/acetone 1 :1 (10g/l; Trans-Blot Transfer Medium, Bio-Rad, Glattbrugg, Switzerland). 0.3 mL of this solution was applied to the sample plate followed by 0.8 mL of sample solution in 10 % ACN/H₂O/2% HCOOH corresponding to 5 % of total sample. After evaporation at room temperature, the sample spot was treated for 10 sec with 10 mL of 2 % HCOOH. Reflectron positive MALDI mass spectra were recorded on a PerSeptive Voyager Elite mass spectrometer (Framingham, MA, USA) at 20 kV accelerating potential in the delayed extraction mode using standard settings for delay times and grid voltages. Samples were irradiated by a nitrogen laser pulse at 337 nm and 256 laser shots were summed into a single mass spectrum. Spectra were in most cases calibrated internally on known background signals. Method

A tryptic mass map is derived from the reflector MALDI mass spectrum generated in the delayed extraction mode . This high resolution mode provides after subtraction of background signals (e.g. trypsin autodigestion products) a list of monoisotopic peptide masses with high accuracy (generally better than 80 ppm).

The list of monoisotopic masses used for the database search is a follows:

828.307 831.343 904.513 912.245

722.054

918.439 933.066 966.409 1169.672 1211.712

1224.662 1244.622 1319.732 1418.742 1564.812

1633.822 1768.002 1776.972 1790.842 1793.922

1827.922 1876.902 1929.912 1951.922 1967.892

1987.002 2149.142 2224.152 2237.982 2464.152

This list is matched against the masses of the predicted tryptic peptides for each entry in the sequence database (GenePept database) together with some restrictions, e.g. regular tryptic fragments, range of molecular masses of parent protein up to 200 kDa, expected mass accuracy better than 50 ppm. Human splicing factor SF3a120 is thus identified by 10 experimental peptide masses matching those expected for the parent protein and only 6 matching peptides for the subsequent search results (Fig. 4). This large gap between the first hit and the following candidates is essential for a reliable identification. Detailed analysis on the basis of the retrieved protein sequence reveals a total of 13 peptides covering 15 % of the amino acid residues of the protein. In this case the peptides are all clustered at the N-terminal part of the protein, which indicates that the analyzed protein is only a fragment. The overall sensitivity of the method is in the low femtomolar range and allows the analysis of Coomassie Blue stained spots of low intensity.

Data base search with tryptic mass list

Data base searches (Peptide Search program from M. Mann, EMBL Heidelberg applied to GenPept database, (Mann, M., In Microcharacterization of Proteins; (1994) Kellner, R., Lottspeich, F., Meyer, H. E., Eds.; VCH: Weinheim, 223-245.) are performed with expected accuracies between 50 and 150 ppm for masses of tryptic peptides depending on type of calibration. Generally a mass deviation of +/- 15 kDa with respect to the molecular mass derived from the respective SDS data is tolerated for the parent protein. The minimum number of peptides required for a match is individually adjusted dependent on extent of background contributions.

Example 8: Cloning and expression of the GDI protein

As outlined above, the sequence data obtained for the polypeptide corresponding to spot 2A, 2B match with the amino acid sequence of human rho GDP-dissociation Inhibitor 2 (in the following abbreviated as GDI). In the following molecular cloning as well as expression of this protein is described. These procedures can likewise be applied to other polypeptides or proteins according to the present invention, identified as outlined above.

(i) Screening of cDNA libraries:

A human Leukemia 5'stretch plus cDNA library in Igtl 1 with mRNA isolated from nontreated Jurkat leukemic T-cells, 5'-extended and oligo(dT) plus random primed (Clontech Inc., CA), is screened with a 63bp primer coding for human GDI-peptide of amino acids 176-197. The fragment is nick translated with a-³²P dCTP (Amersham, UK) and polymerase/DNase (International Biotechnology Inc., CT) prior to screening the cDNA library. Phage isolation is performed according to standard procedures (Sambrook et al., Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press (1989)). cDNA inserts are size estimated by agarose gel electrophoresis after cleavage with EcoR1, are purified with glass beads (BIO 101, CA) and are subcloned into pEMBL vectors. Restriction mapping with endonucleases is performed, and selected fragments are further purified with the Quiagen ion exchange procedure (Diagen, FRG).

(ii) Generation of full-length cDNA: in order to obtain a full-length cDNA, mRNA is isolated from the HL60 cell line which has been deposited at the Deutsche Sammlung von Mikrooorganismen und Zellkulturen (DSM), Braunschweig, FRG, on April 02, 1997 (5x10⁷ cells) with the guanidinium-thiocyanate method and further purified on oligo-dT cellulose spin columns (Pharmacia Biotech, Sweden). 1 mg mRNA is reverse transcribed with 2.5U of avidin myeloblastosis virus reverse transcriptase (Boehringer Mannheim, FRG) using the following 30-mer oligonucleotide primer: (5'-CTCATCATCTTTGTCCATTTCCTGCAGCTC-3'), (SEQ. ID. NO. 10) corresponding to a 5'-stretch of the nucleic acid sequence obtained in (i), above. The cDNA product obtained is purified on a Chroma-Spin 400 column (Clontech Inc., CA) and residual mRNA is hydrolyzed with 0.5M NaOH. The single-stranded cDNA is ligated with an anchor 27-mer oligonucleotide, harboring an EcoR1 restriction site, using T4 RNA ligase (Amplifinder; Clontech Inc., CA). The cDNA obtained is PCR-amplified with Taq polymerase (AmpliTaq, Perkin-Elmer, CA) using a 5'-anchor primer (Clontech Inc., CA) complementary to the ligated 27-mer oligonucleotide, and a 3'-primer (5'-TTTCAGAAGCTTCTGTGGTGG- 3'), (SEQ. ID. NO. 11 ) corresponding to the 5'-upstream sequence of the sequence mentioned above and harboring a Hindlll restriction site. The protocol for PCR amplification used: 35 cycles of 94°C, 45s; 56°C, 45s; 72°C, 1min. The PCR product is size fractionated on 1.5% agarose gel, purified with glass beads and digested with the corresponding restriction endonucleases before ligation into digested pBiuescript vector.

(iii) Sequence analysis:

DNA sequence analysis is carried out with T7 DNA polymerase (Pharmacia Biotech, Sweden) and [a-^SJdATP (Amersham, UK). The entire cDNA is determined by DNA sequence analysis on both strands and the PCR amplified 5'end is analysed in independently cloned fragments. All sequences obtained are analysed with the Wisconsin Package (Sambrook et al., 1989) and compared with the EMBL data bank.

(iv) Protein expression:

The entire coding region for GDI obtained from cDNA screening and the additional fragment coding for the 5' end is subcloned in pGEX2T (Pharmacia) for recombinant GDI expression.

The cDNA fragments are subcloned using oligonucleotide linkers containing BamH1 (5') and EcoR1 (3') sites, enabling a forced orientation into the bacterial expression vector pGEX2T. The pGEX construct is used to transform E. co// strain DH5a. The correct construct is selected by restriction map analysis. The pGEX transformant with the correct insert coding for GDI is cultured in LB broth at 37°C until ODβoo reaches the log phase, at which time the expression of the glutathione S-transferase-G protein fusion protein is induced by addition of 0.1 mM IPTG. Four hours later the induced E. co/;^' are collected by centrifugation and resuspended in 3% of the original culture volume in sonication buffer (20mM Tris, pH 7.4, 50mM NaCI, 1mM DTT, and 4mM MgCI₂). These cells are sonicated for 30sec and centrifuged for 5min at 12000g. A 100ml portion of a 50% slurry of glutathione- agarose in sonication buffer is added to 1.5ml supernatant and tumbled at 4°C for 1 hour to adsorb the fusion proteins. The glutathione-bound protein is washed extensively with sonication buffer and resuspended in 0.5ml of buffer supplemented with 2.5mM CaCI₂. The full length GDI protein is cleaved from the resin-immobilized fusion protein by digestion with thrombin (0.1-0.2 units/ml) for 1hour at room temperature.

The expressed and released GDI protein is analysed on SDS-PAGE with 12% acrylamide, prepared according to U.K. Laemmli, Nature 227 (1970), 680, and protein bands are visualized by Coomassie stain.

Example 9: Polyclonal antibody production (i) Peptide synthesis:

The 16 amino acid GDI-peptide (KTLLGDGPWTDPK), (SEQ. ID. NO. 12) corresponding to amino acids 50-63 of human rho GDP-dissociation Inhibitor 2, is synthesized using a Milligen/Biosearch 9600 peptide synthesizer according to the protocol provided by the manufacturer (MilliGen/Biosearch, Novato, CA). (ii) Synthesis of conjugate:

The GDI-peptide is synthesized with an additional cysteine at the amino terminus and conjugated to either BSA (bovine serum albumin) or KLH (keyhole limpet hemocyanin) by conventional methods with MBS (m-maleimidobenzoyl-Λ/-hydroxysuccinimide ester) as the linker (T. Kitagawa et al., J. Biochem. 79 (1976), 233 ). Briefly described, the carrier protein and sulfo-MBS are suspended in PBS (phosphate-buffered saline (50mM sodium phosphate, 150mM NaCI)), pH 8.5, and mixed at 70rpm for 20min, 25°C The peptide : carrier ratio is 50 : 1 , and the sulfo-MBS to peptide ratio is 2 : 1. Peptide dissolved in PBS, pH 5.0 is added dropwise to derivatized carrier, and the pH is maintained at 7.2 during the 2h/25°C mixing. The conjugated peptide is then dialyzed for 16h/4°C against PBS.

(iii) Polyclonal antibody production:

Two New Zealand White rabbits are each initially injected via lymph node with 300mg of the GDI-peptide KTLLGDGPWTDPK (SEQ. ID. NO. 13) conjugated to KLH in PBS, pH 7.5, emulsified with complete Freund's adjuvant. Subsequent intramuscular injections of 150mg antigen per rabbit emulsified with incomplete Freund's adjuvant are administered every 2 weeks for 16 weeks. Blood samples are collected and tested for the desired antibodies according to known procedures (e.g. J. Sambrook et al., see above). Antibodies of interest are isolated and purified e.g. according to J. Sambrook et al. (see above).

Claims

Claims:

1. A polypeptide selected from the group consisting of :

(1 A.1B) a polypeptide having on a 2D-gel an apparent pi value of 6.4 and an apparent molecular weight of 22 kDa;

(2A.2B) a polypeptide having on a 2D-gel an apparent pi value of 6.8 and an apparent molecular weight of 22 kDa;

(3A) a polypeptide having on a 2D-gel an apparent pi value of 7.6 and an apparent molecular weight of 26 kDa;

(7 A) a polypeptide having on a 2D-gel an apparent pi value of 5.8 and an apparent molecular weight of 27 kDa;

(10A, 6B) a polypeptide having on a 2D-gel an apparent pi value of 5.7 and an apparent molecular weight of 19 kDa;

(11 A) a polypeptide having on a 2D-gel an apparent pi value of 5.7 and an apparent molecular weight of 15 kDa;

(15A) a polypeptide having on a 2D-gel an apparent pi value of 4.0 and an apparent molecular weight of 20 kDa; (16A) a polypeptide having on a 2D-gel an apparent pi value of 7.2 and an apparent molecular weight of >90 kDa;

(4B) a polypeptide having on a 2D-gel an apparent pi value of 7.6 and an apparent molecular weight of 25.5 kDa;

(5B) a polypeptide having on a 2D-gel an apparent pi value of 5.7 and an apparent molecular weight of 17 kDa;

(1 OB) a polypeptide having on a 2D-gel an apparent pi value of 5.5 and an apparent molecular weight of 29 kDa;

(11B) a polypeptide having on a 2D-gel an apparent pi value of 5.3 and an apparent molecular weight of 33 kDa;

(17B) a polypeptide having on a 2D-gel an apparent pi value of 6.2 and an apparent molecular weight of 19 kDa; (18B) a polypeptide having on a 2D-gel an apparent pi value of 7.5 and an apparent molecular weight of 25 kDa; and

2. A pharmaceutical composition comprising a polypeptide as claimed in claim 1.

3. A process for isolating a polypeptide, comprising (a) induction of a cellular condition related to apoptosis in the HL 60 cell line, said cellular condition being achievable by culturing said HL 60 cell line with an appropriate final concentration of staurosporine until a cellular condition related to apoptosis is detectable upon monitoring said cellular condition, (b) identification of a polypeptide showing upon analysis by 2-dimensional polyacrylamide gel electrophoresis, being in one dimension an immobilized pH-gradient electrophoresis and in the other dimension SDS polyacrylamide gel electrophoresis, a differential expression pattern or a differential migration behavior compared with the non-induced cell line and (c) isolation of said thus identified polypeptide.

4. A polypeptide obtainable by the process as claimed in claim 3, the culturing of the HL 60 cell line being for 12 hours and the staurosporine being present in a final concentration of 1 ╬╝M, said polypeptide preferably being selected from the group consisting of:

(2A,2B) a polypeptide having on a 2D-gel an apparent pi value of 6.8 and an apparent molecular weight of 22 kDa;

(7 A) a polypeptide having on a 2D-gel an apparent pi value of 5.8 and an apparent molecular weight of 27 kDa; (8A) a polypeptide having on a 2D-gel an apparent pi value of 5.5 and an apparent molecular weight of 26 kDa;

(17A) a polypeptide having on a 2D-gel an apparent pi value of 4.85 and an apparent molecular weight of >100 kDa; and

(18A) a polypeptide having on a 2D-gei an apparent pi value of 4.9 and an apparent molecular weight of >100 kDa;

5. A polypeptide obtainable by the process as claimed in claim 3, the culturing of the HL

60 cell line being for 10 hours and the staurosporine being present in a final concentration of 0.5╬╝M, said polypeptide preferably being selected from the group consisting of:

(12A, 3B) a polypeptide having on a 2D-gel an apparent pi value of 5.1 and an apparent molecular weight of 5 kDa; (4B) a polypeptide having on a 2D-gel an apparent pi value of 7.6 and an apparent molecular weight of 25.5 kDa;

6. A polypeptide as claimed in any one of claims 1 , 2, 4 or 5 for use as a drug.

7 Use of a polypeptide as claimed in any one of claims 1 , 2, 4 or 5 as a marker or surrogate marker for monitoring a cellular condition achievable by application of staurosporine .

8. Use of a polypeptide claimed in any one of claims 1 , 2, 4 or 5 as a diagnostic marker or surrogate marker for a disease, said disease being selected from the group consisting of diseases responsive to induction of a cellular condition achievable by application of staurosporine, and diseases responsive to inhibition of a cellular condition achievable by application of staurosporine.

9. Use of a polypeptide as claimed in any one of claims 1 , 2, 4 or 5 as a drug discovery target for identification of a drug for treatment of a disease, said disease being selected from the group consisting of diseases responsive to induction of a cellular condition achievable by application of staurosporine, and diseases responsive to inhibition of a cellular condition achievable by application of staurosporine.

10. Use of a polypeptide as claimed in any one of claims 1 , 2, 4 or 5 in the preparation of a pharmaceutical composition for treatment of a disease responsive to induction of a cellular condition achievable by application of staurosporine.

11. Use of a polypeptide as claimed in any one of claims 1 , 2, 4 or 5 in the preparation of a pharmaceutical composition for treatment of a disease responsive to inhibition of a cellular condition achievable by application of staurosporine.

12. The use according to any of the claims 7 to 11 , wherein said cellular condition is a condition related to apoptosis.

13. The use according to any of the claims 8 to 12, wherein said disease responsive to inhibition of a cellular condition achievable by application of staurosporine is selected from the group consisting of neurodegenerative diseases, neuropathies, immunodeficiencies, geriatric diseases and transplantation rejection diseases.

14. The use according to any of the claims 8 to 12, wherein said disease responsive to induction of a cellular condition achievable by application of staurosporine is selected from the group consisting of hyperproliferative diseases, autoimmune diseases and dermatological diseases.

15. A DNA molecule encoding a polypeptide as claimed in any one of claims 1, 2, 4 or 5..

16.. An oligonucleotide capable of specifically binding to a naturally occurring DNA or RNA coding for a polypeptide as claimed in any one of claims 1 , 2, 4 or 5.

17. An antibody directed to a polypeptide as claimed in any one of claims 1 , 2, 4 or 5.

18. The process according to claim 3, wherein said culturing is performed for at least about 3 hours.

19. The process according to claim 18, wherein said culturing is performed for 3 to 18 hours.

20. The process according to claim 3, wherein said two-dimensional polyacrylamide gel electrophoresis is in the first dimension an immobilized pH-gradient electrophoresis and in the second dimension a SDS polyacrylamide gel electrophoresis.