DE19822985C1 - P53 family tumor suppressor genes - Google Patents

Abstract

The invention relates to new tumor suppressor genes of the p53 family, polypeptides that encode them and their use. It preferably relates to nucleic acids encoding KET, in particular rats, humans and mice.

Description

The invention relates to new tumor suppressor genes of the p53 family, polypeptides that they encode and their use. It preferably relates to nucleic acids encoding KET, especially human and mouse.

Genes that play a major role in tumorigenesis can functional mode of action can be roughly classified. Adds a gain-of-function mutation an allele that has an activating effect on tumorigenesis, the affected gene is called Called Oncogen. A loss-of-function mutation is necessary on both alleles (inactivating) to make tumorigenic changes possible, one speaks of a tumor suppressor gene. The most prominent and most studied Tumor suppressor gene codes for the nuclear transcription factor p53 (over 2000 Medline entries in the past year; NCBI database) with the main function in the Control of the cell cycle and apoptosis (Levine 1997). p53 is in human tumors over 50% mutated before (Hollstein et al. 1991), inherited p53 mutations lead to a increased tumor incidence in carriers of the defective allele (Evans and Lozano 1997). There are currently four p53 knock-out mouse lines by several working groups were made independently and serve as animal models for humans. p53-deficient animals (genotype: +/- and - / -) show an increased number at an early age Tumor rate (Donehower et al. 1992, Harvey et al. 1993b). Embryo and organogenesis is, however, generally unremarkable, so that p53 deficient animals do not lose their + / + Wild-type siblings can be distinguished (Donehower et al. 1992). Show however some p53 - / - embryos had a characteristic defect on day 13.5 of embryogenesis in the morphology of the head area, an exencephaly, in which the closure of the Neural tube in the forebrain and midbrain has not occurred. This malformation occurs in 16% of the homozygous-deficient CV129 embryos while only 8% of CV129 × C57BL / 6- p53 - / - hybrid embryos are affected. Interestingly, that also varies Tumor incidence in the CV129 background and CV129 × C57BL / 6 hybrid background. Tumors generally develop faster in CV129 mice than in CV129 × C57BL \ 6 hybrids; in addition, teratomas occur more frequently in CV129 animals (Donehower et al. 1995, Harvey et al. 1993a). Such differences  can only by the different constitution of the genetic background be explained. This shows the existence in the different genetic background differentiated compensation efficiency versus p53 loss and existence of related gene products that demonstrate the function of p53 during embyogenesis and Perform cancerogenesis.

Oncogene (1997) 15, 1363-1367 describes the rat KET cDNA with SEQ ID No. 1 (deposited with EMBL, Gen Bank and DDJB) and its amino acid sequence known.

The invention had for its object to provide further genes for proteins encode which play a role in the control of the cell cycle and apoptosis.

The invention is based on the finding that the protein KET is so remarkable Homology in its amino acid sequence to p53 was found to be p53 in one p53 family can be summarized. Like p53, KET has a transactivation, a DNA binding and an oligomerization domain. The highest level of homology can be found in the DNA binding domain. Between KET and p53 it is 75%. The The coding cDNAs were isolated from the rat.

According to the invention, the human KET cDNA (SEQ ID No. 2) was cloned; an alignment of the derived amino acid sequence (SEQ ID No 3) with that from rat and the sequences of human p53 and p73 is shown in Fig. 1. The rat KET amino acid sequence shows 98% homology to that of humans.

A chromosomal localization of the gene was found on chromosome 3q in humans and 16 in mice (cf. Fig. 2 Genetic mapping), from which it can be concluded that KET functions as a tumor suppressor. Interestingly, the mouse ket gene maps to an area that is deleted in the early stages of pancreatic carcinogenesis and presumably contains an angiogenesis suppressor, Loh2 (gene symbol: Loh2), for which Ket is therefore a candidate.

According to the invention, it was found that the KET protein in tumor suppression is involved. Of particular interest were those tumors in which previously no changes in the p53 wild type allele were described. The chromosomal Localization of the responsible  Tumor suppressor genes can be determined by cytogenetic analysis, the loss of heterozygosity Identify (LOH) areas to be predicted. It was found that the KET / Ket gene in humans or mice mapped and detected in such LOH regions.

According to the invention, the KET / Ket gene was also mapped in humans and mice flanking markers (Figure 2). For precise chromosomal localization in humans radiation hybrids (Radiation Hybrids; GeneBridge 4 Panel, Research Genetics) used, the mapping in the mouse was carried out in a M. musculus × M. spretus Backcross generation. The ket gene mapped between the somatostatin gene and the Apolipoprotein D gene on Chr. 3q of humans. The same gene order was made in Chr. 16 of the mouse confirmed. A (left) chromosomal section of human chromosome 3q with the position of the KET locus; B (middle) position of the ket locus on Chr. 16 of the mouse. C. (right) Ket haplotypes and marker genes on Chr. 16. Each column represents two Haplotypes from Chr. 16, the number of backcross individuals is given below (upper Number: filled squares / rectangles SEG / 1 allele; empty squares / rectangles C57BL6J allele; lower number: the opposite) Gene symbols: CKCN2 / Clc2 - chloride channel 2; SST / Smst - somatostatin; KET / Ket - p53 related protein KET; APOD / Apod - apolipoprotein D; GAP43 / Gap43 - growth affecting protein 43.

The invention therefore relates to the KET nucleic acids - KET cDNA of man and mouse - the SEQ ID No. 2 (human KET cDNA).

Furthermore, all T can be replaced by U (ribonucleic acid).

The invention furthermore relates to the polypeptides for which the cDNAs code, preferably the human KET sequence SEQ ID No. 3rd  

The production takes place according to known methods, such as. B. by Isolation and sequencing from cDNA libraries.

Furthermore, the invention relates to the use of the KET nucleic acids and Polypeptides as a basis for the development of specific and effective Cancerostatics. They are used to build genes and vectors that the basis for the development of these pharmaceutically relevant substances represent.

They are also used to develop diagnostic kits, such as: B. for Predict cancer risk. The subject of this is also diagnostic Test kits.

Furthermore, genomic ketone clones were characterized Human and mouse through the differential representation of intron spanning PCR fragments. These PCR tests were also made for the Identification of Ket-positive BACs (Bacterial artificial chromosome) in one genomic BAC library (Genome Systems Inc) used. These BACs contain DNA from the CV129 / J mouse strain, so that subfragments of the BAC Clones can be used directly to construct the ket targeting vector. On the first 5 'exons were also found in the BACs identified so far. From the Human and rat full-length KET cDNA sequence were Primer sequences derived from mouse exon 1 specific PCR tests to lead.

Ket-deficient mice were also produced.

The production of mice bearing zero alleles of the ket gene required four successive processes:

• - Isolation and characterization of a suitable section of the target gene.
• - Cloning of a targeting vector in which the open reading frame of the Target genes through the integration of a selection marker (complete Transcription unit for the neomycin resistance gene) is destroyed.
• - Homologous integration of the recombination construct into the genome of embryonic stem cells and subsequent selection for antibiotics Resistance.  
• - Injection of ESCs in blastocysts (or coculture with morulae) and subsequent uterine transfer.

Targeting vectors are also an issue. In an embodiment variant the existing BAC after digestion with suitable ones Restriction endonucleases subcloned into pBluescript or pUC (optimal size of the subclones 5-10 kb). Via further restriction mapping and STS mapping (Sequence Tagged sites) using PCR and / or Southern blotting was a subclone contig created, which can be viewed as a fine mapping of the 5 'gene region. The human and rat cDNA information was used for this. As a general rule the open reading frame can be interrupted in two places: Es became the first translated exons in one case and the whole in the other putative DNA binding domain switched off. To the tissue-specific Expression of ket during embryo and organogenesis in chimeras and To track ket-deficient mice, a β-galactosidase Cloned cassette so that it is under the control of the ket promoter. In general, two forms of targeted gene targeting were possible: At Using an insertion vector, the complete vector (a Crossover event was required) for the more common one Replacement vector only integrated part of the choice of intragener Restriction interfaces was dependent (two crossover events required).

Gene deactivation in embryonic stem cells (ESCs)

As already mentioned above, the genomic BAC library used is CV 129 origin. Most of this mouse stem also became common ESCs isolated. The advantage of using isogenic material lies in the higher probability of homologous recombination. ESCs were checked after transfection (electroporation) by G418 selection. in the In the case of the replacement vector, an internal vector was added Thymidine kinase cassette for negative selection (Gancyclovir) in non-homologous Integration used.

Successful homologous recombination was confirmed by DNA analysis (Southern Blot) checked.

By injecting such modified ESCs into blastocysts, too Embryonic chimeras are made and diagnosed.  

For blastocyst injection or morula aggregation only genotyped ESCs used. Chimeric pre-implantation embryos were placed in the Uterine horns transmitted from sham pregnant recipient mice. Chimeras were in test pairs on a successful germline transmission from ESC Descendants checked. F1 / F2 descendants of chimeras that zero the ket Carrying alleles either heterozygous or homozygous were determined using Southern blot or genotyped PCR analyzes.

Furthermore, the invention is closer by an example and a sequence listing explained.

example Obtaining the human KET cDNA (SEQ ID No. 2)

For the production of human KET cDNA, 1 × 10 ⁶ clones of a human skeletal muscle cDNA library (Stratagene) were checked with samples which originated from a rat KET cDNA (Schmale and Bamberger, 1997). A single positive clone, hu41m, was obtained and the 3226 bp insert was sequenced in two directions using vector specific and internal primers. The insert contained an open reading frame of 1360 bp, homologous to the N-terminus of the rat KET sequence, but the coding sequence was interrupted by an unknown sequence according to the QQHQHLLQ motif at position 448. Examination of 6 × 10 ⁵ clones of a human keratinocyte cDNA library (Clontech) with a sample derived from the 3 'end of hu41m revealed two overlapping clones, hu6k and hu10k, which contained part of the cDNA clone hu41 were identical and extended the sequence towards the 3 'end. To obtain 3 'end sequences, the EST database with the 3' untranslated region of the rat KET clone was searched. After finding several homologous EST clones, two of them (IMAGE Consortium clone ID 149663 and 137665) were completely sequenced. For the amplification and direct sequencing of a 1.2 Kb fragment from the human skin cDNA, PCR primers according to the 3 'end of the cDNA clone hu10k and the 5' end of the EST clone 149663 were used. The complete cDNA contains 4846 bp, including 27 bp of the most likely truncated 5 'untranslated region and 2776 bp of the 3' untranslated region. To demonstrate the adjacent arrangement of the sequences obtained from different sources, PCR primers positioned according to the translation start and stop codons were used for the amplification of the complete protein coding sequences of the KET of human skin cDNA. The cDNA contains an open reading frame that codes for 680 amino acids ( Fig. 1). The putative start methionine is preceded by a translation stop codon (not shown) in the grid. A comparison of the amino acid sequences of the KET of humans and rats shows a 98% identity ( Fig. 1). This remarkable interspecific preservation of KET proteins extends over the entire molecular length. It is even more pronounced in the middle part, which contains the DNA binding area; 248 amino acids are completely unchanged. The KET proteins are far more conserved than the corresponding p53 proteins from humans and rats, which are 79% homologous. Their identity only reaches 91% in the DNA binding area. Human p73 shows a total of 58% identity with human KET. Conservation is again highest in the DNA binding region with an identity of 86%, while the N-terminal region, with the exception of the transactivation region, deviates the most. In addition to the transactivation and DNA binding areas, p53, p73 and KET share a well-preserved oligomerization area. The three proteins are likely to be able to form mixed oligomers that have specific biological functions.

For proteins that do not tolerate any change for functional reasons can, such as those that have multiple binding sites Amino acid sequences are generally as well preserved as in KET dated Humans and rats can be observed. This preservation leaves suspect that KET can be an evolutionary old gene, which is likely in the development and differentiation of higher invertebrates and vertebrates was included in the general basic functions. p53 can later change from have developed its precursor gene as a protein that is specific for Functions such as monitoring genome damage is responsible. The scope of the genotoxic load depends on physiological factors and Environmental factors that, at least in part, for the different species are different. So the relative diversity of p53, in comparison to KET, which reflect the species-specific requirements for such a system.  

For the mapping process based on PCR, STS from Humans (hKET8) and two KET STS of mice (muKET8 and muKET)) amplified (see Table 1).

The primer positions were defined in such a way that fragments were formed which contain an intron flanked by exon sequences. This made it possible to identify correct PCR fragments by comparing them with the known rat KET cDNA sequence. Intron-containing PCR fragments were chosen specifically for mapping the ket gene in mice in order to obtain easily detectable fragment-length polymorphisms after restriction with suitable endonucleases. The exon-intron boundaries were derived by comparing the KET amino acid sequence with that of p53 and p73 (Schmale and Bamberger, 1997; Kaghad et al. 1997). The exon sequences corresponded to the human KET amino acid sequence ( Fig. 1) as follows: hKET9, amino acid residues 360-390; muKET8, amino acid residues 360-400; muKET9, amino acid residues 383-438. PCRs were carried out as already described (Lengeling et al. 1995). Nucleotide sequences of primers for the Mit microsatellite marker D16Mit57 were obtained from the MIT mouse genome database. Mouse segregation data were processed with the GENE-LINK computer program (Montagutelli, 1990). hKET8, comprising Intron 8, was mapped using the GeneBridge 4 radiation hybrid mapping panels (Research Genetics, Huntsville, AL). This panel represents 91 radiation hybrid clones of the entire human genome. The KET gene was mapped on the human chromosome 3q27 between the microsatellite markers D3S1580 and D3S1314 ( Fig. 2A). The most likely gene order and spacing were D3S1580 - 2.2 cR - WI 6145 - 7.1 cR - KET - 4.7 cR - WI1189 - 8.9 cR - D3S1314. This area is flanked by somatostatin, SST (O'Hara et al. 1988) and apolipoprotein D, APOD (Warden et al. 1992). Information about the chromosomal localization of SST and APOD and the gene order was taken from the chromosome 3 map (San Antonio Genome Center; http://genome.uthcsa.edu/Maps/frame.html).

3q27 is the middle part of a region of a well-documented syntenia to mouse chromosome 16, which extends from CLCN2 to GAP43 and Clc2 to Gap43 in the mouse genome (DeBry and Seldin, 1996; Lengeling et al. 1995). To determine whether the mouse ket locus falls within the homologous region, the ket gene was mapped using an interspecies backcross of the mouse (C57BL / 6J wrl + xSEG / 1 + / +) ^* F ₁ wrl / + x ( C57BL / 6J wrl +), which was originally established for mapping the wobbler gene (Kaupmann et al. 1992). This interspecies backcrossing panel was characterized for over 150 loci that were distributed across all autosomes and the X chromosomes. An improved map of chromosome 16 has been created for mapping the chloride channel gene Clc2 (Lengeling et al. 1995). Both mouse KET-PCR fragments provided informative restriction fragment length variants (RFLVs) which were used for the segregation analysis. The fragment with intron 8 (muKET8) was cut with Msp1, muKET9 with Rsa1. KET was discovered between Smst and D16Mit63 with lodserves <8 ( Fig. 2B, C). The mouse homologue of human apolipoprotein D, Apod, the gene marker on human Chr 3q closely linked distally to KET, was not mapped in the M. musculus × M. Spretus backcrossing panel, but detailed mapping data are available (Reeves and Cabin, 1997; Warden et al. 1992; Reeves et al. 1997). D16Mit 57 was mapped distal to Apod (Reeves and Cabin, 1997) and uses a fragment length polymorphism between the C57BL / 6J muscle (111 bp) and the spretus SEG / 1 muscle (135 bp). No recombination of ket and D16Mit57 was found in 50 meiosis. On the back crossing of M. musculus × M. spretus, the most likely gene sequences and distances were Cen - D16Mit87 - 3.9 ± 1.7 cM - Smst, D16Mit102 - 4 ± 2.77 cM - Ket, D16Mit 57 - 14 ± 4 , 91 cM - D16 with 63.

The loss of the long arm of chromosome 3 is rarely found (cf. Chitayat et al. 1996). The symptoms with eliminations 3q27 → qter differ significantly and are not sufficient to identify a specific syndrome to derive. While in two cases only minor facial abnormalities, Delays in development and hypotension have been reported other serious, multiple congenital abnormalities, including Anophthalmia and cerebral atrophy.

In some human cancer tissues (e.g., oesophageal cancer and squamous Carcinomas), LOH areas were found on Chr 3 containing 3q27 (Sato et al. 1994; Wang et al. 1996). Although statistically significant, it was a loss of 3q, which compared to other chromosomal abnormalities with a relatively low frequency occurred in these tumors.  

Comparative mapping data showed areas of a fully preserved one Syntenia between Chr 3q and mouse chromosomes 3, 9 and 16 (see DeBry and Seldin, 1996), two of which are the predicted suppressor genes Loh1 and Loh2 harbors that at distinct levels of tumor development in one transgenic mouse model of islet cell carcinoma (Dietrich et al. 1994; Parangi et al. 1995; Shi et al. 1997) can be deleted. The ket gene falls in the same LOH Area with Loh2 (LOH about 15 cM, 14-29 cM from Cen, flanked by the Microsatellite markers D16Mit35 and D16Mit39; (see Parangi et al. 1995). Of Loh2 is believed to be an angiogenesis suppressor (Parangi et al. 1995). In fact, the p53 protein has been shown to be the Angiogenesis in fibroblasts indirectly inhibited by the positive regulation of the Thrombospondin-1 expression (Dameron et al. 1994). Therefore, Ket is a candidate for Loh2 by its chromosomal localization and by its putative Function.

Table 1

PCR primer for STS from the KET / Ket regions

Cited references

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SEQUENCE LOG

Claims (15)

1. KET-coding nucleic acid, characterized by the human KET cDNA with the sequence SEQ ID No. 2nd 2. KET nucleic acids according to claim 1, characterized in that T by U is exchanged. 3. KET nucleic acids according to one of claims 1 or 2, characterized in that it is completely complementary. 4. polypeptides for which KET nucleic acids encode according to one of claims 1 to 3. 5. Polypeptides according to claim 4 characterized by the human KET sequence SEQ ID No. 3rd 6. Vectors containing a DNA encoding a KET nucleic acid or polypeptides of claims 1 to 5 included. 7. Host cells containing the vectors according to claim 6. 8. Use of KET nucleic acid or polypeptides according to one of claims 1 to 5 for the detection of KET nucleic acids in biological samples. 9. Use according to claim 8, characterized in that a biological Sample with at least one compound of SEQ ID No. 2 and / or SEQ ID No. 3, if necessary brings into contact with a carrier molecule according to conventional methods and the Detection based on the hybridization complex formed by physical or chemical methods.   10. Use according to claim 8 or 9, characterized in that the nucleic acid Is DNA that may contain a homozygotic deletion. 11. Use according to claim 8, characterized in that the nucleic acid is RNA. 12. Use according to one of claims 8 to 11, characterized in that the Nucleic acid is labeled, preferably by a radioisotope, a bioluminescent, a chemiluminescent or fluorescent compound, a metal chelate, or a Enzyme. 13. Use according to one of claims 8 to 12, characterized in that the biological sample tumor tissue with angiogenesis of humans or the Mouse is. 14. Use of KET nucleic acids according to one of claims 1 to 3 for detection the presence or absence of the human chromosome 3q27 or its Fragments based on the hybridization product between chromosomal DNA and the KET nucleic acid. 15. Use of KET nucleic acids according to one of claims 1 to 3 for detection the presence or absence of mouse chromosome 16 or its fragments based on the hybridization product between chromosomal DNA and the KET Nucleic acid.