DE19818619A1 - New nucleic acid sequences expressed in bladder tumor tissue, and derived polypeptides, for treatment of bladder tumor and identification of therapeutic agents - Google Patents

Abstract

Polypeptide fragment sequences (I) are new and comprise 55 open reading frame amino acid sequences given in the specification, that are highly expressed in bladder tumor. Independent claims are also included for the following: (1) a nucleic acid (II) encoding (I) selected from 50 nucleic acid sequences given in the specification, or its allelic variants or antisense sequences; (2) bacterial or phage artificial chromosomes and cosmid clones containing functional genes, or the chromosome region containing them, corresponding to (II) for use as gene transfer vehicles; (3) antisense sequences to (II); (4) an expression cassette (III) comprising (II), or their fragments, plus control and regulatory sequences; (5) DNA fragments (IV containing a gene isolated using (II); (6) host cells (V) containing (II) as a heterologous component; (7) recombinant production of (I), or their fragments, by culturing cells of (7); (8) an antibody (VI) directed against (I), or its fragments, encoded by (II); (9) 56 partial polypeptide sequences, all given in the specification, and sequences having at least 80% homology with them;

Description

The invention relates to human nucleic acid sequences from bladder tumor tissue, which code for gene products or parts thereof, their functional genes, the at least encode a biologically active polypeptide and its use.

The invention further relates to the polypeptides and obtained via the sequences their use.

One of the main causes of cancer death is the bladder tumor, new ones to fight it Therapies are necessary. Therapies used so far, such as B. chemotherapy, Hormone therapy or surgical removal of the tumor tissue often does not result to complete healing.

The phenomenon of cancer is often associated with overexpression or underexpression Genes in the degenerate cells, although it is still unclear whether they changed Expression rates are the cause or consequence of the malignant transformation. The Identification of such genes would be an essential step in the development of new ones Cancer therapies. The spontaneous onset of cancer often goes a variety ahead of mutations. These can have various effects on the Have expression patterns in the affected tissue, e.g. B. sub or Overexpression, but also expression of shortened genes. Several such Changes caused by such cascade mutations can eventually lead to malignant ones Lead to degeneracies. The complexity of such relationships makes it difficult experimental approach very much.

For the search for candidate genes, d. H. Genes compared to tumor tissue are more strongly expressed in normal tissue, a database is used that consists of so-called ESTs. ESTs (Expressed Sequence Tags) are sequences of cDNAs, d. H. reverse transcribed mRNAs, the molecules that express the expression of Reflect genes. The EST sequences are for normal and degenerate tissues determined. Such databases are used by various operators e.g. T. commercial offered. The ESTs of the LifeSeq database used here are usually between 150 and 350 nucleotides in length. They represent one for a particular gene distinctive pattern, although this gene is usually much longer (<2000 Nucleotides). By comparing the expression patterns of normal and tumor tissue ESTs can be identified that are important for tumor development and proliferation are. However, there is the following problem: Because of different constructions of the cDNA libraries found EST sequences to different regions of an unknown gene would result in a completely in such a case incorrect ratio of the occurrence of these ESTs in the respective tissue. This would only be noticed when the full gene is known and thus the ESTs can be assigned to the same gene.

It has now been found that this possibility of error can be reduced if all ESTs from the respective tissue type are assembled beforehand before the expression patterns are compared with one another. Thus were overlapping ESTs of the same gene in longer sequences summarized (s. Fig. 1, Fig. 2a and Fig. 3). By extending this and thus covering a much larger gene range in each of the respective banks, the error described above should be largely avoided. Since there were no existing software products for this purpose, programs for assembling genomic sections were used, which were used in a modified form and supplemented by own programs. A flowchart of the assembly procedure is shown in Fig. 2b1-2b4.

The nucleic acid sequences Seq. ID No. 1 50 can be found as Candidate genes play a role in bladder cancer.

The nucleic acid sequences Seq are of particular interest. ID Nos. 2-5, 7-13, 16, 18, 20, 23, 26-27, 31-32, 36, 45.

The invention thus relates to nucleic acid sequences encoding a gene product or a part thereof, comprising

• a) a nucleic acid sequence selected from the group of Nucleic acid sequences Seq. ID Nos. 2-5, 7-13, 16, 18, 20, 23, 26-27, 31-32, 36, 45.
• b) an allelic variation of the nucleic acid sequences mentioned under a)
  or
• c) a nucleic acid sequence which is complementary to that under a) or b) is called nucleic acid sequences.

The invention further relates to a nucleic acid sequence according to one of the sequences Seq. ID Nos. 2-5, 7-13, 16, 18, 20, 23, 26-27, 31-32, 36, 45 or a complementary one or allelic variant thereof and the nucleic acid sequences thereof, the one Have 90% to 95% homology to a human nucleic acid sequence.

The invention also relates to the nucleic acid sequences Seq. ID No. 1 to Seq. ID No. 50, which are increased expressed in the bladder tumor tissue.

The invention further relates to nucleic acid sequences comprising part of the above called nucleic acid sequences, in such a sufficient size that they with the sequences Seq. ID Nos. Hybridize 1-50.

The nucleic acid sequences according to the invention generally have a length from at least 50 to 4500 bp, preferably a length of at least 150 to 4000 bp, in particular a length of 450 to 3500 bp.

With the partial sequences Seq. ID Nos. 1-50 can according to more common Process practice also expression cassettes are constructed, being on the Cassette of at least one of the nucleic acid sequences according to the invention together with at least one control or regulatory sequence, such as B. a suitable promoter is combined. The Sequences according to the invention can be inserted in sense or antisense orientation his.

The literature contains a large number of expression cassettes or vectors and Promoters known that can be used.  

Expression cassettes or vectors are to be understood: 1. bacterial, such as. B., phagescript, pBs, ϕX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pnH18a, pNH46a (Stratagene), pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), 2. eukaryotic, such as B. pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia).

Suitable promoters are to be understood as control or regulatory sequences. Two preferred vectors are the pKK232-8 and the PCM7 vector. In particular the following promoters are meant: lacI, lacZ, T3, T7, gpt, lambda P _R, trc, CMV, HSV thymidine kinase, SV40, LTRs from retrovirus, and mouse metallothionein-I.

The DNA sequences on the expression cassette can be a Encode fusion protein, which is a known protein and a biologically active Polypeptide fragment.

The expression cassettes are also the subject of the present invention.

The nucleic acid fragments according to the invention can be used to produce Full length genes can be used. The genes available are also a subject of the present invention.

The invention also relates to the use of the nucleic acid Sequences, as well as the gene fragments available from the use.

The nucleic acid sequences according to the invention can be used with suitable vectors in Host cells are brought in, in which as a heterologous part the on the nucleic acid Genetic information contained fragments that is expressed.

The host cells containing the nucleic acid fragments are also a subject of the present invention.

Suitable host cells are e.g. B. prokaryotic cell systems such as E. coli or eukaryotic cell systems such as animal or human cells or yeast.

The nucleic acid sequences according to the invention can be in sense or antisense Shape can be used.

The polypeptides or their fragments are produced by cultivating the Host cells according to common cultivation methods and subsequent isolation and Purification of the peptides or fragments, also using conventional methods. The invention further relates to nucleic acid sequences that have at least one partial sequence encode a biologically active polypeptide.

Furthermore, the present invention relates to partial polypeptide sequences, so-called ORF (open reading frame) peptides, according to the sequence protocols ORF ID Nos. 51-106.

The invention further relates to the polypeptide sequences that have at least one 80% Homology, in particular 90% homology to those according to the invention Partial polypeptide sequences of the ORF. ID Nos. 51-106.  

The invention also relates to antibodies against a polypeptide or fragment thereof are directed, which of the nucleic acids of the sequences according to the invention Seq. ID No. 1 to Seq. ID 50 can be encoded.

Antibodies include, in particular, monoclonal and phage display antibodies understand.

The polypeptides according to the invention of the sequences ORF ID Nos. 51-106 can too used as a tool to find active substances against the bladder tumor, which is also the subject of the present invention.

The present invention also relates to the use of Nucleic acid sequences according to the sequences Seq. ID No. 1-50 for expression of Polypeptides used as tools to find active substances against the bladder tumor can be used.

The invention also relates to the use of the polypeptide partial sequences found ORF. ID No. 51-106 as a drug in gene therapy to treat the Bladder tumor, or for the manufacture of a medicament for the treatment of the Bladder tumor.

The invention also relates to pharmaceuticals which have at least one polypeptide partial sequence ORF. ID No. 51-106 included.

The nucleic acid sequences according to the invention found can also be genomic or mRNA sequences.

The invention also relates to genomic genes, their exon and intron structure and their Splice variants, available from the cDNAs of the sequences Seq. ID No. 1-50, as well their use together with suitable regulatory elements, such as suitable ones Promoters and / or enhancers.

With the nucleic acids (cDNA sequences) according to the invention Seq. ID No. 1-50 genomic BAC, PAC and cosmid libraries are screened and specifically human clones are isolated via complementary base pairing (hybridization). The BAC, PAC and cosmid clones isolated in this way are hybridized with the aid of fluorescence in situ hybridization to metaphase chromosomes and corresponding chromosome sections on which the corresponding genomic genes lie are identified. BAC, PAC and cosmid clones are sequenced in order to elucidate the corresponding genomic genes in their complete structure (promoters, enhancers, silencers, exons and introns). BAC, PAC and cosmid clones can be used as independent molecules for gene transfer (see FIG. 5).

The invention also relates to BAC, PAC and cosmid clones containing functional genes and their chromosomal location, according to the sequences Seq. ID. No. 1 to Seq. ID No. 50, for use as a vehicle for gene transfer.  

Meanings of technical terms and abbreviations

Nucleic acids = nucleic acids are to be understood in the present invention: mRNA, partial cDNA, full length cDNA and genomic genes (chromosomes).
ORF = Open Reading Frame, a defined sequence of amino acids that can be derived from the cDNA sequence.
Contig = a set of DNA sequences that can be combined into one sequence due to their very similarities (consensus)
Singleton = a contig that contains only one sequence.

Explanation of the alignment parameters

minimal initial match = minimal initial identity range
maximum pads per read = maximum number of insertions
maximum percent mismatch = maximum deviation in%.

Explanation of the pictures

Fig. 1 shows the systematic gene search in the Incyte LifeSeq database.

Fig. 2a shows the principle of the EST assembly

Fig. 2b1-2b4 shows the overall principle of the EST assembly

Fig. 3 shows the in silico subtraction of gene expression in various tissues

FIG. 4a shows the determination of tissue-specific expression on electronic Northern.

FIG. 4b shows the electronic Northern

Figure 5 shows the isolation of BAC and PAC genomic clones.

The following examples illustrate the preparation of the invention Nucleic acid sequences without the invention on these examples and Restrict nucleic acid sequences.

example 1 Search for tumor-related candidate genes

First, all ESTs of the corresponding tissue from the LifeSeq-Da tenbank (from October 1997) extracted. These were then created using the program GAP4 of the Staden package with the parameters 0% mismatch, 8 pads per read and assembled a minimum match of 20. Which is not in the GAP4 database Recorded sequences (failures) were first mismatched at 1% and then again assembled at 2% mismatch with the database. From the contigs of the database, the consisted of more than one sequence, consensus sequences were calculated. The Singletons of the database, which consisted of only one sequence, were not with the sequences added to the GAP4 database again at 2% mismatch assembled. Again, the consensus sequences were determined for the contigs. All other ESTs were reassembled at 4% mismatch. The consensus sequences were extracted again and with the previous consensus sequences as well as the Finally, singletons and the sequences not included in the database assembled at 4% mismatch. The consensus sequences were formed and with the Singletons and Fails used as a basis for the tissue comparisons. By this procedure could be ensured that among the parameters used all sequences represented mutually independent gene regions.

Fig. 2b1-2b4 illustrates the extension of the bladder tissue ESTs.

The sequences of the respective tissues assembled in this way were then compared using the same program ( FIG. 3). For this purpose, all sequences of the first tissue were first entered in the database. (It was therefore important that they were independent of each other.)

Then all sequences of the second tissue were compared to all of the first. The results were sequences that were specific for the first and the second tissue were, as well as those that occurred in both. In the latter, the ratio of Frequency of occurrence in the respective tissues evaluated. All that Evaluation of the assembled sequences, programs themselves developed.

All sequences that occurred more than four times in one of the compared tissues as well as all that occurred at least five times as frequently in one of the two tissues were examined further. These sequences were subjected to an electronic Northern (s. Example 2.1), whereby the distribution in all tumor and normal tissues was examined (s. Fig. 4a and Fig. 4b). The relevant candidates were then extended using all Incyte ESTs and all ESTs in public databases (see Example 3). The sequences and their translation into possible proteins were then compared with all nucleotide and protein databases and examined for possible regions coding for proteins.

Example 2 Algorithm for the identification and extension of partial cDNA sequences with a changed expression pattern

An algorithm for finding over- or underexpressed genes will be explained below. For the sake of clarity, the individual steps are also summarized in a flow chart (see FIG. 4b).

2.1 Electronic Northern blot

To a partial DNA sequence S, e.g. B. a single EST or a contig of ESTs are created using a standard homology search program, e.g. B. BLAST (Altschul, S.F., Gish W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) J. Mol. Biol., 215, 403-410), BLAST2 (Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Nucleic Acids Research 25 3389-3402) or FASTA (Pearson, W.R. and Lipman, D.J. (1988) Proc. Natl. Acad. Sci. USA 85 2444-2448), the homologous sequences in different ordered by tissues (private or public) EST libraries. The determined thereby (relative or absolute) tissue-specific occurrence frequencies of these Partial sequence S are referred to as electronic Northern blot.

2.1.1

Analogous to the procedure described under 2.1, the sequence Seq. ID No. 16 found 17.7 times stronger in normal bladder tumor tissue than in normal Bladder tissue occurs.

The result is as follows:

Electronic Northern for SEQ. ID. NO: 16

The following Northerns were also found in an analogous procedure:

Electronic Northern for SEQ. ID. NO: 8

Electronic Northern for SEQ. ID. NO: 2

Electronic Northern for SEQ. ID. NO: 3

Electronic Northern for SEQ. ID. NO: 4

Electronic Northern for SEQ. ID. NO: 5

Electronic Northern for SEQ. ID. NO: 6

Electronic Northern for SEQ. ID. NO: 7

Electronic Northern for SEQ. ID. NO: 8

Electronic Northern for SEQ. ID. NO: 9

Electronic Northern for SEQ. ID. NO: 10

Electronic Northern for SEQ. ID. NO: 11

Electronic Northern for SEQ. ID. NO: 12

Electronic Northern for SEQ. ID. NO: 13

Electronic Northern for SEQ. ID. NO: 14

Electronic Northern for SEQ. ID. NO: 15

Electronic Northern for SEQ. ID. NO: 17

Electronic Northern for SEQ. ID. NO: 18

Electronic Northern for SEQ. ID. NO: 19

Electronic Northern for SEQ. ID. NO: 20

Electronic Northern for SEQ. ID. NO: 21

Electronic Northern for SEQ. ID. NO: 22

Electronic Northern for SEQ. ID. NO: 23

Electronic Northern for SEQ. ID. NO: 24

Electronic Northern for SEQ. ID. NO: 25

Electronic Northern for SEQ. ID. NO: 26

Electronic Northern for SEQ. ID. NO: 27

Electronic Northern for SEQ. ID. NO: 28

Electronic Northern for SEQ. ID. NO: 29

Electronic Northern for SEQ. ID. NO: 30

Electronic Northern for SEQ. ID. NO: 31

Electronic Northern for SEQ. ID. NO: 32

Electronic Northern for SEQ. ID. NO: 33

Electronic Northern for SEQ. ID. NO: 34

Electronic Northern for SEQ. ID. NO: 35

Electronic Northern for SEQ. ID. NO: 36

Electronic Northern for SEQ. ID. NO: 37

Electronic Northern for SEQ. ID. NO: 38

Electronic Northern for SEQ. ID. NO: 39

Electronic Northern for SEQ. ID. NO: 40

Electronic Northern for SEQ. ID. NO: 41

Electronic Northern for SEQ. ID. NO: 42

Electronic Northern for SEQ. ID. NO: 43

Electronic Northern for SEQ. ID. NO: 44

Electronic Northern for SEQ. ID. NO: 45

Electronic Northern for SEQ. ID. NO: 46

Electronic Northern for SEQ. ID. NO: 47

Electronic Northern for SEQ. ID. NO: 48

Electronic Northern for SEQ. ID. NO: 49

Electronic Northern for SEQ. ID. NO: 50

2.2 Fisher test

To decide whether a partial sequence S of a gene in a library for Normal tissue occurs significantly more or less often than in a library for degenerate tissue becomes Fisher's Exact Test, a standard statistical procedure (Hays, W.L., (1991) Statistics, Harcourt Brace College Publishers, Fort Worth), carried out.

The null hypothesis is: the two libraries can increase in frequency S homologous sequences cannot be distinguished. If the null hypothesis with can be rejected with sufficient certainty, the gene belonging to S is considered interesting candidate for a cancer gene is accepted and it will be the next step tries to extend its sequence.

Example 3 Automatic extension of the partial sequence

The automatic extension of the partial sequence S takes place in three steps:

• 1. Determination of all sequences homologous to S from the total amount of the Available sequences with the help of BLAST
• 2. Assembly of these sequences using the standard program GAP4 (Bonfield, J.K., Smith, K.F., and Staden R. (1995), Nucleic Acids Research 23 4992-4999) (Contig formation).
• 3. Calculation of a consensus sequence C from the assembled sequences.

The consensus sequence C will generally be longer than the starting sequence s. Your Northern electronic blot will therefore differ from that for S. Another Fisher test decides whether the alternative hypothesis of deviation from uniform expression can be maintained in both libraries. If this is the case, an attempt is made to extend C in the same way as S. This iteration is continued with the respectively obtained consensus sequences C _i (i: index of the iteration) until the alternative hypothesis is rejected (if H ₀ exit; termination criterion I) or until automatic extension is no longer possible (while C _i < C _i-1 ; termination criterion II).

In the case of termination criterion II, you get the one after the last iteration present consensus sequence a complete or approximately complete sequence of a gene associated with cancer with high statistical certainty can be brought.

Analogously to the examples described above, those described in Table I could Nucleic acid sequences from bladder tumor tissue can be found.  

Furthermore, the peptide sequences could be used for the individual nucleic acid sequences (ORF's) that are listed in Table II, with few No peptide can be assigned to nucleic acid sequences and some Nucleic acid sequences can be assigned to more than one peptide. As before mentioned above are both the nucleic acid sequences as well as the Peptide sequences assigned to nucleic acid sequences are the subject of present invention.  

TABLE II

The inventive nucleic acid sequences Seq. ID No. 1 to Seq. ID No. 50 of determined candidate genes and the determined amino acid sequences Seq. ID No. 51-106 are described in the sequence listing below.

Sequence listing

Claims (39)

1. A nucleic acid sequence encoding a gene product or a part thereof

• a) a nucleic acid sequence selected from the group Seq. ID No. 2-5, 7-13, 16, 18, 20, 23, 26-27, 31-32, 36, 45.
• b) an allelic variation of the nucleic acid sequences mentioned under a)
  or
• c) a nucleic acid sequence which is complementary to the nucleic acid sequences mentioned under a) or b).

2. A nucleic acid sequence according to one of the sequences Seq. ID Nos. 1-50, or a complementary or allelic variant of it. 3. Nucleic acid sequence Seq. ID No. 1 to Seq. ID No. 50, characterized, that they are expressed increased in bladder tumor tissue. 4. BAC, PAC and cosmid clones containing functional genes and their chromosomal Localization, according to the sequences Seq. ID. No. 1 to Seq. ID No. 50, for Use as a vehicle for gene transfer. 5. A nucleic acid sequence according to claims 1 to 4, thereby characterized in that they have 90% homology to a human nucleic acid Sequence. 6. A nucleic acid sequence according to claims 1 to 4, characterized characterized in that they have 95% homology to a human nucleic acid Sequence. 7. A nucleic acid sequence comprising a part of that in claims 1 to 6 called nucleic acid sequences, in such a sufficient size that they hybridize with the sequences according to claims 1 to 6. 8. A nucleic acid sequence according to claims 1 to 7, characterized characterized in that the size of the fragment has a length of at least 50 to 4500 bp.   9. A nucleic acid sequence according to claims 1 to 7, characterized characterized in that the size of the fragment has a length of at least 50 to 4000 bp. 10. A nucleic acid sequence according to any one of claims 1 to 9, the at least encodes a partial sequence of a biologically active polypeptide. 11. An expression cassette comprising a nucleic acid fragment or a Sequence according to one of claims 1 to 9, together with at least one Control or regulatory sequence. 12. An expression cassette comprising a nucleic acid fragment or a The sequence of claim 11, wherein the control or regulatory sequence is one is a suitable promoter. 13. An expression cassette according to one of claims 11 and 12, characterized characterized in that the DNA sequences on the cassette Encode fusion protein, which is a known protein and a biologically active Polypeptide fragment. 14. Use of the nucleic acid sequences according to claims 1 to 10 for Production of full length genes. 15. A DNA fragment comprising a gene resulting from use according to claim 14 is available. 16. Host cell containing as a heterologous part of its expressible genetic Information a nucleic acid fragment according to one of claims 1 to 10. 17. Host cell according to claim 16, characterized in that it is a is prokaryotic or eukaryotic cell system. 18. Host cell according to one of claims 16 or 17, characterized in that the prokaryotic cell system E. coli and the eukaryotic cell system animal, human or yeast cell system. 19. A method of making a polypeptide or fragment, thereby characterized in that the host cells are cultivated according to claims 16 to 18 become.   20. An antibody directed against a polypeptide or fragment which from the nucleic acids of the sequences Seq. ID No. 1 to Seq. ID No. 50 encoded that is obtainable according to claim 19. 21. An antibody according to claim 20, characterized in that it is monoclonal is. 22. An antibody according to claim 20, characterized in that it is a phage Display antibody is. 23. partial polypeptide sequences, according to the sequences Seq. ID Nos. ORF 51-106. 24. polypeptide partial sequences according to claim 22, with at least 80% homology to these sequences. 25. A polypeptide derived from a phage display which is attached to the Can bind partial polypeptide sequences according to claim 24. 26. polypeptide partial sequences according to claim 22, with at least 90% homology to these sequences. 27. Use of the partial polypeptide sequences according to the sequences Seq. ID No. 51-106, as tools for finding active substances against the bladder tumor. 28. Use of the nucleic acid sequences according to the sequences Seq. ID No. 1 to Seq. ID No. 50 for expression of polypeptides used as tools for detection of agents against the bladder tumor can be used. 29. Use of the nucleic acid sequences Seq. ID No. 1 to Seq. ID No. 50 in sense or antisense form. 30. Use of the polypeptide partial sequences Seq. ID No. 51-106 as a drug in gene therapy to treat the bladder tumor. 31. Use of the polypeptide partial sequences Seq. ID No. 51-106, for manufacturing a medicine to treat the bladder tumor. 32. Medicament containing at least one polypeptide partial sequence Seq. ID No. 51-106. 33. A nucleic acid sequence according to claims 1 to 10, thereby characterized as being a genomic sequence.   34. A nucleic acid sequence according to claims 1 to 10, thereby characterized that it is an mRNA sequence. 35. Genomic genes, their promoters, enhancers, silencers, exon structure, Intron structure and its splice variants, obtainable from the cDNAs of the sequences Seq. ID No. 1 to Seq. ID No. 50. 36. Use of the genomic genes according to claim 33, together with appropriate regulatory elements. 37. Use according to claim 34, characterized in that the regulative Element is a suitable promoter and / or enhancer. 38. A nucleic acid sequence according to claims 1 to 7, thereby characterized in that the size of the fragment has a length of at least 300 to 3500 bp.