EP0612355A1 - Detection de la proteine-kinase humaine p58 et de son adn pour le diagnostic de certains cancers - Google Patents

Detection de la proteine-kinase humaine p58 et de son adn pour le diagnostic de certains cancers

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Publication number
EP0612355A1
EP0612355A1 EP92921986A EP92921986A EP0612355A1 EP 0612355 A1 EP0612355 A1 EP 0612355A1 EP 92921986 A EP92921986 A EP 92921986A EP 92921986 A EP92921986 A EP 92921986A EP 0612355 A1 EP0612355 A1 EP 0612355A1
Authority
EP
European Patent Office
Prior art keywords
gene
protein kinase
human
absence
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP92921986A
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German (de)
English (en)
Other versions
EP0612355A4 (fr
Inventor
Vincent J. Kidd
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UAB Research Foundation
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UAB Research Foundation
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Filing date
Publication date
Application filed by UAB Research Foundation filed Critical UAB Research Foundation
Publication of EP0612355A1 publication Critical patent/EP0612355A1/fr
Publication of EP0612355A4 publication Critical patent/EP0612355A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57515—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the breast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates to a method and to a kit for detecting a genetic mutation associated with a cancer and, more particularly, to a method and a kit using a labelled probe to detect a gene whose absence is associated with certain types of cancers.
  • the invention further relates to a method and to a kit for detecting a genetic mutation associated with a cancer using a labelled antibody to detect a protein whose absence is associated with certain types of cancers.
  • the protein kinase p34 cdc2 has been shown to be a crucial component of a normal mitotic promoting factor complex, which includes other protein components such as the cyclin regulatory subunits.
  • the p34 cdc2 protein kinase has been shown to be required for both the G 2 - to S-phase transition in the cell cycle.
  • p34 cdc2 protein kinase is the catalytic subunit of starfish, clam, and Xenopus oocyte maturation-promoting factor (MPF) (Arion et al., 1988; Draetta et al., 1988; Dunphy et al., 1988; Gutier et al., 1988) and its somatic cell mitotic counterpart (Draetta and Beach, 1988; Langan et al., 1989; Brizuela et al., 1989).
  • MPF Xenopus oocyte maturation-promoting factor
  • p58 gene is well conserved evolutionarily, that its expression is regulated during murine embryogenesis, and that its activity is coordinately regulated with p34 cdc2 during the cell cycle (Kidd et al, 1991; Xiang et al., in preparation for publication).
  • the p34 cdc2 protein kinase has been localized to human chromosome 10 (Spurr et al., 1988). However, the locations of the other members of the human CDC- related protein kinase gene family have to date remained unknown. It was postulated that knowledge of the locations of these cell cycle genes on human chromosomes may provide important information regarding the possible mechanisms that underlie cellular
  • a human cyclin-related gene (potentially a cell cycle regulatory subunit) corresponds to the translocation breakpoint for the bell-linked candidate oncogene or human chromosome 11 (Motokura et al, 1991).
  • a human cyclin A gene which is a known cell cycle regulatory subunit, was identified as a locus interrupted by hepatitis B virus integration associated with hepatocellular carcinoma (Wang et al, 1990).
  • the protein p58 cdc2 exhibits a 68% homology to the human p34 cdc2 protein.
  • the p58 protein is 436 amino acids in size, is apparently regulated by phosphorylation and Ca 2+ calmodulin, and is localized to the cytoplasm and nucleus of both rapidly dividing and differentiated cells. Expression of the corresponding mRNA is regulated during the cell cycle, peaking during S-phase. Elevated expression of the CDC-related kinase in eukaryotic cells leads to an apparent block of the cell cycle at late telophase and abnormal cytokinesis.
  • the expression of the p58 gene has been found to be regulated via sequences within its 3' untranslated region and protein results in apparent changes in DNA replication. It was also shown that a severalfold increase in the expression of the p58 kinase alters normal progression of cells through the cell cycle and dramatically increases the frequency of mitotic abnormalities. It was suggested that alterations in the level of endogenous p58 can affect the cell cycle, with too little p58 allowing cells to proceed through the cell cycle more rapidly, whereas too much p58 inhibits cell cycle progression. The results were viewed as being consistent with the possibility that p58 kinase acts as a negative regulator of some component of the cell cycle pathway. It was concluded that the altered cell cycle phenotype and mitotic abnormalities associated with these overexpressors suggest that elevated expression of the p58 kinase interferes with the late events associated with normal mitosis.
  • Yet another object of the present invention is to provide a method for diagnosing cancers associated with the absence of the gene encoding human p58 protein which can be carried out easily by a technician using, for example, a pre-prepared kit.
  • FISH fluorescent in situ hybridization
  • PCR nested polymerase chain reaction
  • the present invention relates to a method of diagnosing a cancer associated with the absence of the gene encoding human p58 protein comprising the steps of;
  • (c) determining the presence or absence the of gene on chromosome 1. Included among the cancers associated with the absence of the gene encoding human p58 protein are neuroblastoma, ductal carcinoma of the breast, malignant melanoma, Merkel cell carcinoma or endocrine neoplasia.
  • the present invention relates to a method for diagnosing cancer comprising reacting cells with an antibody directed to the p58 protein kinase and detecting the presence or absence of specific binding of the antibody to the cells.
  • kits for diagnosing a cancer associated with the absence of the gene encoding human p58 protein comprising a labelled probe including a DNA sequence complementary to the human p58 protein kinase gene or to a portion of the p58 protein kinase gene which is long enough to identify such gene, and a solution suitable for obtaining hybridization of the probe to a number 1 human chromosome.
  • kits for diagnosing a cancer associated with the absence of the gene encoding human p58 protein kinase gene comprising an antibody directed to human p58 protein kinase and a means of detecting the presence or absence of specific binding of the antibody to the p58 protein kinase.
  • Figure 1 depicts restriction endonuclease digestions of human p58 chromosomal gene cosmid 2A.
  • One microgram of cosmid DNA was digested with the indicated enzyme, electrophoresed on a 1 % agarose-TAE gel, and Southern transferred. The blot was then hybridized with a 1.5-kb EcoRI fragment from the cDNA containing the entire p58 protein kinase open reading frame (ORF).
  • the arrows on the left indicate BamHI fragments of interest 6.5 and 0.8 kb in size.
  • the sizes of lambda HindIII molecular weight markers are shown on the right;
  • Figure 2 depicts a human-mouse somatic cell hybrid analyses.
  • Total genomic DNA (12 ⁇ l) was digested with either BamHI (A) or EcoRI (B), electrophoresed on 0.8% agarose-TAE gels, and Southern transferred. The blots were then hybridized with the same p58 cDNA probe as used in Fig. 1.
  • the lanes correspond to the following DNAs: lane 1, GM/NA09925; lane 2, GM/NA09926; lane 3, GM/NA09927; lane 4, GM/NA09928; lane 5, GM/NA09929; lane 6, GM/NA09930A; lane 7, GM/NA09931; lane 8, GM/NA09932; lane 9, GM/NA09933; lane 10, GM/NA09934; lane 11,
  • the arrows on the right of the panels indicate fragments of interest.
  • the lambda Hindm molecular weight size markers are indicated on the left;
  • Figure 3 depicts the hybridization of the human p58 protein kinase gene to human metaphase chromosomes
  • a human chromosome 1- specif ⁇ c centromere probe was also included in the reaction. In situ hybridization and detection were performed as described by Lichter and colleagues.
  • the arrows indicate the p58 hybridizing regions detected by biotin-labelled p58 cosmid 2A.
  • [B] The same human metaphase chromosomes spread shown in A after Giemsa handing. The arrows indicate the position of the biotin-labelled p58 cosmid 2A sequences shown in A on the same Giemsa-handed chromosomes;
  • Figure 4 depicts human chromosome 1 idiogram showing localization of the p58 protein kinase gene.
  • the arrow on the left shows the location of all signals in all in situ metaphase spreads using the p58 cosmid 2A as a probe.
  • the arrows on the right demark the location of the microdissection points for the nested PCR amplification experiment;
  • Figure 5 depicts localization of the p58 protein kinase gene on human chromosome 1.
  • Nested PCR amplification was performed using primers specific for the p58 protein kinase cosmid 2A gene. Templates for the reactions were as follows: lane 1, microdissected chromosome 1 fragment corresponding to 1 pter; lane 2, microdissected chromosome 1 fragment corresponding to 1 qter: lane 3, negative control containing no DNA template: lane 4, total human genomic DNA.
  • MW indicates the 123-bp molecular weight standard ladder. Sizes of the MW standards are given on the left, while the size of the expected p58 gene product (342bp) is denoted on the right.
  • the present inventors have now isolated and localized the chromosomal gene encoding p58 protein to the human gene map.
  • the gene contains 13 exons and 12 introns, spans approximately 12 kb of DNA, and is localized to the telomeric end of human chromosome 1, lp36.
  • This region of chromosome 1 is commonly deleted in numerous tumors that are neural crest in origen, including malignant melanoma and neuroblastoma. The phenotype of these tumors suggests that the gene deleted in these tumors normally suppresses tumor formation, much like the retinoblastoma gene product and the p53 oncoprotein.
  • CDC-related protein kinase gene The isolation of this CDC-related protein kinase gene allowed us to test the hypothesis that this gene is involved in neuroblastoma and malignant melanoma.
  • the p58 cosmid clone containing the corresponding gene from human chromosome lp36, is used in fluorescent in situ hybridization (FISH) studies with a control cosmid for a single copy sequence on the long arm, lq, to determine the frequency of p58 gene deletion in these tumors.
  • FISH fluorescent in situ hybridization
  • DNA from the same chromosome preparations can be examined for mutations in the p58 coding region by polymerase chain reaction (PCR) amplification of the DNA and subsequent DNA sequence analysis of the various exonic regions.
  • PCR polymerase chain reaction
  • the human chromosomal gene corresponding to the previously reported 3.5-kb p58 mRNA was isolated from a human chromosomal DNA cosmid library.
  • the corresponding human p58 cDNA has been reported and its sequence is known (Bunnell et al, 1990a).
  • the murine-human somatic cell panel number 1 and murine, human, and Chinese hamster control DNAs were obtained from the NIGMS Human Genetic Mutant Cell Repository. Restriction endonuclease digestions were performed according to manufacturer's specifications and Southern blot analysis was performed as previously described (Bunnell et al., 1990a,b).
  • DNA probes were labeled by random oligonucleotide primer incorporation of [ ⁇ - 32 P]dCTP (Amersham, > 3000 Ci/mmol) as described previously (Kidd et al., 1991). In situ Hybridization
  • Amplification and chromosome staining were performed as specified by Oncor.
  • Photographs were taken using a Leitz fluorescent microscope and Ektachrome 800/1600 film. Slides were then destained and Giemsa banded according to Cannizzaro and Emanuel (1984).
  • the nested PCR primers were generated based on the position of intron/exon junctions surrounding nucleotides 527 to 804 of the human p58 cDNA
  • Primers 3 and 4 are nested inside the region amplified by primers 1 and 2.
  • a portion of the sequences of primers 1 and 3 is derived from the adjacent intronic sequence of the p58 chromosomal gene. Inclusion of intronic DNA sequences in the PCR probe design will also help to ensure that this p58 locus is, indeed, the gene being analyzed and limits the possibility of interference from the related p58 sequence on human chromosome 15. Chromosome microdissection was performed on a Giemsa-banded human metaphase chromosome spread as described previously with some modifications (Han et al, 1991). Freshly prepared metaphase chromosome spreads were baked at 80 °C for 30 min and G- banded using standard cytogenetic technique.
  • Chromosome microdissection was performed on an Olympus microscope under x750 magnification with a long working distance objective (x50).
  • the microscope has a rotable stage so that the best cutting angle can easily be obtained.
  • the dissected chromosome fragments were delivered into 50 ⁇ l of PCR reaction mixture [67 mM Tris-HCl, pH 8.8, 6.7 mM MgCl 2 , 170 ⁇ l/ml BSA, 16.6 mM (NH 4 ) 2 SO 4 , 10% DMSO, 0.02 mM dNTP, and 100 ng each of the primers].
  • Primers 1 and 2 were used for the first round PCR of 20 cycles (94°C 1 min, 50°C 2 min.
  • the 0.192 kb BamHI fragment corresponds to a 0.192 bp BamHI fragment found in the coding region of the reported human cDNA (Bunnel et al., 1990a), plus an additional 75-bp intron found in this region, and indicates that this portion of the chromosomal gene is interrupted by a small intron.
  • the observed cosmid restriction fragments of 6.5 and 0.8 kb are highlighted since they clearly segregate with a single chromosome locus on the somatic cell panel.
  • This human cosmid p58 genomic locus has been sequenced and found to contain exons that are absolutely identical to the reported human p58 cDNA (Eipers, Lahti, and Kidd, in preparation; Bunnell et al., 1990a).
  • restriction fragments 21, 6.5, 4.1, and 0.8 kb were observed (Fig. 2A). Only the 21-kb BamHI fragment is not found in the BamHI digest of the p58 cosmid isolate shown in Fig. 1.
  • the 6.5-, 4.1-, and 0,8-kb BamHI fragments are all found in human-mouse somatic cell hybrids containing human chromosome 1 (Fig. 2A, lanes 1, 2, 3 and 9; Table 1), while the 21-kb BamHI fragment segregates with a separate locus corresponding to human chromosome 15 (Fig. 2A, lanes 1, 2, 3, 4, 9, 10, 13, and 18; Table 1), The 0.192-kb BamHI fragment is too small to be observed and resolve reliably by Southern blot analysis of total genomic DNA. However, its presence was verified by other techniques (see below).
  • the apparent 6.5-kb BamHI band in lane 6 of Fig. 2A is an anomaly, most likely due to incomplete digestion of DNA.
  • the 4.1- and 0.8-kb BamHI fragments are not concomitantly observed in this lane.
  • chromosome 1 (0% discordance)
  • a highly related p58 sequence appears to be localized to human chromosome 15 (0% discordance).
  • chromosome 1 near a telomere with no significant background in 20 metaphase spreads analyzed by FISH (Fig. 3 and 4). In fact, 78 of 80 sister chromatids showed
  • the present inventors After preparing the metaphase cells and screening them with a labelled probe, such as in the preferred embodiment described above, it is determined whether a hybridization occurs. If such hybridization does not occur, then it will be known that the cell is potentially cancerous. More specifically, the present inventors have now found that there are certain tumors associated with the absence of the human p58 protein kinase gene from the number 1 chromosome and that these tumors may be detected using this correlation and appropriately labeled oligonucleotide probes complementary to the gene (or a sufficiently long segment of the gene) or labelled antibodies generated from the protein kinase or, at the very least, the antigenic determinative portion thereof.
  • cancers which may be detected in accordance with the present invention are neuroblastomas, ductal carcinomas of the breast, malignant melanomas, Merkel cell carcinomas or endocrine neoplasias.
  • Labelled antibodies are prepared in accordance with techniques known in the art. Such antibodies may be prepared from protein purified to homogeneity from a natural source or, alternatively, may be obtained recombinantly. Generation of the antibodies from the naturally occurring protein is described in Bunnell et al PNAS 87, 1990. With respect to recombinantly produced protein, it will be appreciated that for the purposes of generating antibodies, it is only necessary that the transformed host, such as E. Coli express the antigenic determinative portion of the protein. Indeed, the present inventors have found that incorporation of the entirety of the p58 kinase gene into an E. Coli host results in the expression of a product which is difficult to recover.
  • the present invention further encompasses a transformed host wherein only the antigenic determinative portion of p58 kinase is inserted into an appropriate host.
  • the antigenic determinative portion of the protein has been found to correspond to a region of the p58 protein from amino acid 1 to 74.
  • the actual preparation of vectors to include the antigenic determinative portion of the protein as well as the transformation of a suitable host were carried out in accordance with techniques well known in the art.
  • kits including either probes or antibodies as well as other reagents required for diagnosis of a patient.
  • Such kits including pre-prepared probe or antibody in combination with other reagents would facilitate application of the method such that a technician with minimal training would be able to cany out the diagnosis.
  • a kit is contemplated including a probe as described above, optionally a label such as biotin for detection with fluorescently labelled avidin, and the reagents necessary to prepare a metaphase cells for screening.

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  • Health & Medical Sciences (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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  • Wood Science & Technology (AREA)
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Abstract

Est décrite une méthode pour diagnostiquer des cancers associés à l'absence du gène encodant la protéine humaine p58, tels que des neuroblastomes, des carcinomes dendritiques du sein, des mélanomes malins, des carcinomes cellulaires de Merkel ou des néoplasies endocriniennes. Dans un mode de réalisation, des cellules, notamment en métaphase, sont préparées à partir d'un tissu examiné. Il est ensuite réalisé une hybridation in situ avec une sonde marquée renfermant une séquence d'ADN qui est complémentaire du gène de la protéine-kinase p58 ou d'une partie dudit gène suffisamment longue pour identifier ce dernier, s'il est présent. La détermination de la présence ou de l'absence du gène sur le chromosome numéro 1 indique qu'une cellule est cancéreuse ou, à tout le moins, potentiellement cancéreuse. Dans un deuxième mode de réalisation, des anticorps spécifiques de la protéine-kinase p58 sont employés pour sa détection. Sont également décrites des trousses renfermant une sonde ou un anticorps tels que précités.
EP92921986A 1991-10-10 1992-10-13 Detection de la proteine-kinase humaine p58 et de son adn pour le diagnostic de certains cancers. Withdrawn EP0612355A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US77430391A 1991-10-10 1991-10-10
US774303 1991-10-10
PCT/US1992/008550 WO1993007297A1 (fr) 1991-10-10 1992-10-13 Detection de la proteine-kinase humaine p58 et de son adn pour le diagnostic de certains cancers

Publications (2)

Publication Number Publication Date
EP0612355A1 true EP0612355A1 (fr) 1994-08-31
EP0612355A4 EP0612355A4 (fr) 1995-06-28

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Application Number Title Priority Date Filing Date
EP92921986A Withdrawn EP0612355A4 (fr) 1991-10-10 1992-10-13 Detection de la proteine-kinase humaine p58 et de son adn pour le diagnostic de certains cancers.

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EP (1) EP0612355A4 (fr)
JP (1) JPH07500496A (fr)
CA (1) CA2120329A1 (fr)
WO (1) WO1993007297A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1298010A (zh) * 1999-11-30 2001-06-06 上海博容基因开发有限公司 一种新的多肽——蛋白激酶38和编码这种多肽的多核苷酸
CN1333351A (zh) * 2000-07-07 2002-01-30 上海博德基因开发有限公司 一种新的多肽——人蛋白激酶27.17和编码这种多肽的多核苷酸

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
No further relevant documents disclosed *
See also references of WO9307297A1 *

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JPH07500496A (ja) 1995-01-19
WO1993007297A1 (fr) 1993-04-15
CA2120329A1 (fr) 1993-04-15
EP0612355A4 (fr) 1995-06-28

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