EP0267275A1 - SEQUENCE ADN CODANT L'ONCOGENE $i(ROS), POLYPEPTIDES CODES A PARTIR DE CELLE-CI, ET DIAGNOSTIC ET AUTRES PROCEDES BASES SUR CETTE SEQUENCE - Google Patents
SEQUENCE ADN CODANT L'ONCOGENE $i(ROS), POLYPEPTIDES CODES A PARTIR DE CELLE-CI, ET DIAGNOSTIC ET AUTRES PROCEDES BASES SUR CETTE SEQUENCEInfo
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- EP0267275A1 EP0267275A1 EP87903664A EP87903664A EP0267275A1 EP 0267275 A1 EP0267275 A1 EP 0267275A1 EP 87903664 A EP87903664 A EP 87903664A EP 87903664 A EP87903664 A EP 87903664A EP 0267275 A1 EP0267275 A1 EP 0267275A1
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- Prior art keywords
- tumor
- detecting
- marker
- oncogene
- polypeptide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/82—Translation products from oncogenes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- 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
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- 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
- C12Q2600/158—Expression markers
Definitions
- RNA tumor viruses which are genes capable of altering the growth properties of normal cells, were first detected as the homologs of transforming genes of RNA tumor viruses (3). Additional cellular oncogenes have been discovered by their amplification in certain tumor cells (12,22) and by DNA transfer techniques (24,14,7,1). The most commonly used assay for detecting oncogenes following DNA transfer has been focus formation in NIH3T3 cells (24). An alternate assay for oncogenes based on tumorigenicity in nude mice is described herein (4,8). The details of this procedure have been published previously (8). In brief, DNA isolated from a tumor is cotransfected into NIH3T3 cells in the presence of a G418 antibiotic resistance gene (33,26). Cells which have taken up foreign DNA are selected by their resistance to G418, grown into colonies, pooled and injected into animals.
- MCF-7-1 Three "pri mary” tumors, called MCF-7-1, MCF-7-2 and MCF-7-3 were obtained. DNA from each of these tumors was capable of efficiently inducing "secondary" tumors following another round of cotransformation into NIH3T3 cells and tumorigenicity assays.
- DNA from all tumors derived from MCF-7-2 DNA contained a common gene which was called mcf2; and DNA from MCF-7-3 and its derived tumors all contained a gene which was called mcf3.
- the isolation of portions of mcf2 and mcf3 was previously described.
- the molecular characterization of mcf3 is herein described.
- mcf3 derives in part from the closest human homolog of the avian v-ros oncogene which is called rosl.
- the human rosl gene appears to- have been activated during gene transfer.
- the nucleotide sequence of the activated gene is also described.
- the human rosl gene like the v-ros gene from the avian sarcoma virus UR2 (17), encodes a-putative transmembrane protein kinase, possibly a growth factor receptor.
- the human v-rosl gene is oncogenic, and that the polypeptide encoded by the human v-rosl gene, which functions as a tyrosine-activated protein kinase and has a truncated extracellular segment, is expressed in human tumor cells.
- the invention concerns a DNA sequence comprising an activated oncogene which encodes a polypeptide capable of transforming NIH3T3 cells and of inducing a tumor when injected into nude mice, and further said polypeptide having tyrosine-specific protein kinase activity, said DNA sequence having a nucleotide sequence substantially as shown in Figure 1.
- This invention also concerns methods for detecting tumor cells which comprise isolating genomic DNA or RNA from a cell, contacting the DNA or RNA so isolated with a detectable marker which binds specifically to at least a portion of the sequence encoding an activated oncogene of this invention, or at least a portion of the RNA sequence encoded by an activated oncogene of this invention, and detecting the marker so bound, the presence of bound marker indicating a predisposition of the subject to the disease.
- This invention also concerns a method of determining the predisposition of a subject to a disease, which comprises isolating the genomic DNA or RNA from a cell from the subject, contacting the DNA or RNA so isolated with a detectable marker which specifically binds to at least a portion of an activated oncogene of this invention, or to a portion of the RNA encoded by an activated oncogene of this invention, and detecting the marker so bound, the presence of bound marker indicating a predisposition of the subject to the disease.
- the invention also concerns a polypeptide molecule encoded by an activated oncogene, said polypeptide having the properties of transforming NIH3T3 cells, inducing a tumor when injected into nude mice, acting as a tyrosine-specific protein kinase, and further said polypeptide having an amino acid sequence substantially as shown in Figure 1.
- Tumor cells and tumors expressing the polypeptide of this invention may be detected with a detectable marker which specifically binds to at least a portion of the polypeptides of this invention. Further, subjects predisposed to diseases associated with the polypeptides of this invention may be identified with a detectable marker which specifically binds to at least a portion .of the polypeptide.
- Methods for detecting a tumor or tumor cells comprise isolating serum from a subject, contacting the serum with a detectable marker which binds specifically to at least a portion of a polypeptide of this invention to form a marker-polypeptide complex and detecting the marker so bound, the presence of bound marker indicating the presence of a tumor.
- the invention concerns a method for treating in a subject a tumor induced by an activated rosl oncogene which comprises isolating an immunoglobulin molecule which specifically binds at least a portion of a polypeptide encoded by the activated rosl oncogene, attaching to the immunoglobulin molecule so isolated a substance which substantially limits the growth of a tumor or which destroys tumors to produce an antitumor immunoglobulin molecule, and contacting the tumor with an effective amount of the antitumor immunoglobulin molecule so produced, thereby limiting tumor growth or destroying the tumor.
- the nucleotide sequence of the common region of the mcf3 cDNA clones is depicted. Below the nucleotide sequence, the predicted amino acid sequence is shown. The numbers at the end of each line refer to the position of the predicted amino acid residues, with position + 1 defining the first amino acid of the mcf3 cDNA which is encoded by the rosl derived part of the locus. The potential membrane spanning domain of 21 hydrophobic amino acids is boxed. The position of two splice junctions close to the point of rearrangement are indicted by arrowheads.
- a DNA sequence comprising an activated oncogene has been isolated which encodes a polypeptide capable of transforming NIH3T3 cells and of inducing a tumor when injected into nude mice.
- the polypeptide also has tyrosinespecific protein kinase activity.
- the DNA sequence has a nucleotide sequence substantially as shown in Figure 4.
- the DNA sequence of this invention encodes a ros oncogene, preferably a rosl gene.
- the sequence may be isolated from a variety of sources, although the presently preferred sequence encodes the human rosl gene.
- the polypeptide produced by the transcription of the gene and the translation of the gene product will vary with the initial DNA sequence.
- a method of detecting a tumor cell which contains the DNA sequence of this invention comprises isolating genomic DNA from a cell, contacting the DNA isolated from the cell with a detectable marker which binds specifically to at least a portion of the DNA sequence of this invention which encodes an activated oncogene and detecting the marker so bound.
- the presence of bound marker indicates the presence of a tumor cell.
- tumor cells which may be detected by the method of the present invention are those arising from astrocytoma and glioblastoma cells.
- the detectable marker may be a labelled DNA sequence, including a labelled cDNA sequ-ehce, having a nucleotide sequence complementary to at least a portion of the DNA sequence of this invention, produced by methods known to those skilled in the subject art.
- the detectable marker may also be a labelled ribonucleotide (RNA) sequence having a nucleotide sequence complementary to at least a portion of the DNA sequence of this invention, and may be isolated by methods known to those skilled in the art.
- RNA ribonucleotide
- Detectable markers of this invention will be labelled with commonly employed radioactive labels, i.e. 32 P, although other labels may be employed.
- the markers will be detected by autoradiographic, spectrophotometric or other means known in the art.
- RNA encoded by a DNA sequence of this invention is described.
- the method comprises isolating RNA from a cell, contacting the RNA isolated from the cell with a detectable marker which binds specifically to at least a portion of the RNA encoded by an activated oncogene and detecting the marker so bound.
- the presence of bound marker indicates the presence of a tumor cell.
- tumor cells which may be detected by the method of the present invention are those arising from astrocytoma and glioblastoma cells.
- the detectable marker may be a labelled DNA sequence, including a labelled cDNA sequence, having a nucleotide sequence complementary to at least a portion of the DNA sequence of this invention, produced by methods known to those skilled in the subject art.
- the detectable marker may also be a labelled ribonucleotide (RNA) sequence haying a nucleotide sequence complementary to at least a portion of the DNA sequence of this invention, and may be isolated by methods known to those skilled in the art.
- RNA ribonucleotide
- Detectable markers of this invention will be labelle with commonly employed radioa&tive labels, i.e. 32 P, although other labels may be employed.
- the markers will be detected by autoradiographic, spectrophotometric or other means known in the art. Methods to be used for the isolation of DNA or RNA for the practice of this invention are well known in the subject art (cf. ref. 13).
- a method of determining the predisposition of a subject to a disease associated with a DNA sequence of this invention involves isolating the genomic DNA of a cell from the subject, contacting the DNA so isolated with a detectable marker which specifically binds to at least a portion of an activated oncogene of this invention and detecting the marker so bound.
- the presence of bound marker indicates a predisposition of the subject to the disease.
- diseases to which a predisposition of a subject may be determined by the method of the present invention are those which arise from human tumor cell lines, especially those of astrocytoma and glioblastoma cells.
- the detectable marker may be a labelled DNA sequence, including a labelled cDNA sequence, having a nucleotide sequence complementary to at least a portion of the DNA sequence of this invention, produced by methods known to those skilled in the subject art.
- the detectable marker may also be a labelled ribonucleotide (RNA) sequence having a nucleotide sequence complementary to at least a portion of the DNA sequence of this invention, and may be isolated by methods known to those skilled in the art.
- RNA ribonucleotide
- Detectable markers of this invention will be labelled with commonly employed radioactive labels, i.e. 32 P, although other labels may be. employed.
- the markers will be detected by autoradiographic, spectrophotometric or other means known in the art.
- a method of determining the predisposition of a subject to a disease comprises isolating the RNA from a cell from the subject, contacting the RNA so isolated with a detectable marker which specifically binds to at least a portion of the RNA encoded by an activated oncogene of this invention and detecting the marker so bound, the presence of bound marker indicating a predisposition of the subject to the disease.
- diseases to which a predisposition of a subject may be determined by the method of the present invention are those which arise from human tumor cell lines, especially those of astrocytoma and glioblastoma cells.
- the detectable marker may be a labelled DNA sequence, including a labelled cDNA sequence, having a nucleotide sequence complementary to at least a portion of the DNA sequence of this invention, produced by methods known to those skilled in the subject art.
- the detectable marker may also be a labelled ribonucleotide (RNA) sequence having a nucleotide sequence complementary to at least a portion of the DNA sequence of this invention, and may be isolated by methods known to those skilled in the art.
- RNA ribonucleotide
- Detectable markers of this invention will be labelled with commonly employed radioactive labels, i.e. 32 P, although other labels may be employed.
- the markers will be detected by autoradiographic, spectrophotometric or other means known in the art.
- a polypeptide molecule encoded by an activated oncogene is also provided by this invention.
- the polypeptide has the properties of transforming NIH3T3 cells, inducing a tumor when injected into nude mice and of acting as a tyrosinespecific protein kinase.
- the polypeptide has an amino acid sequence substantially as shown in Figure 1.
- the presently preferred polypeptide will be encoded by a ros oncogene, preferably by the rosl oncogene, and will be expressed in a human, although the polypeptide may be expressed in a variety of other organisms.
- the polypeptide of this invention may be obtained by synthetic means, i.e. chemical synthesis of the polypeptide from its component amino acids, by methods known to those skilled in the art.
- the polypeptide may be obtained by isolating it from cells expressing the rosl gene, i.e. a cloned rosl gene in a bacterial cell, or by in vitro translation of the mRNA encoded by the rosl gene to produce the polypeptide of this invention. Techniques for the isolation of polypeptides by these means are well known to those skilled in the art.
- a method of detecting a tumor cell involves isolating a cell, contacting the cell with a detectable marker which binds specifically to at least a portion of a polypeptide of this invention and detecting the presence of marker bound to the cell.
- the presence of marker so bound indicates that the cell may be a tumor cell.
- tumor cells which may be detected by the method of the present invention are those arising from astrocytoma and glioblastoma cells .
- the detectable marker will preferably be a labelled immunoglobulin molecule, although other markers known in the art may be employed.
- the immunoglobulin molecule may be an antibody produced by contacting the immune system of an animal with at least a portion of the polypeptide of this invention, or with a synthetic amino acid sequence substantially similar to a portion of a polypeptide of this invention.
- the antibody molecule may also be produced by a combination of recombinant DNA techniques with other techniques known in the art.
- Detectable markers of this invention will be commonly employed markers such as heavy metals, radioactive, e.g. 35S, fluorescent, e.g. fluoresceln, or enzymatic, e.g. peroxidase. Detection of the labelled markers may be carried out by autoradiographic, spectrophotometric, or colorimetric techniques or by other methods known in the art.
- a method for detecting a tumor in. a subject comprises contacting the tumor with a detectable marker which specifically binds at least a portion of a polypeptide of this invention and detecting the marker so bound.
- the presence of bound marker indicates .the presence of a tumor.
- tumors which may be detected by the method of the present invention are those which arise from astrocytoma and glioblastoma cells.
- a presently preferred method comprises introducing into the bloodstream of the patient a detectable amount of the marker such that the marker contacts and binds to a tumor expressing the polypeptide encoded by an activated oncogene, the tumor being detectable thereby.
- the presently preferred marker is a labelled immunoglobulin molecule although other markers known in the art may be employed.
- the immunoglobulin molecule may be an antibody produced by contacting the immune system of an animal with at least a portion of the polypeptide of this invention, or with a synthetic amino acid sequence substantially similar to a portion of a polypeptide of this invention.
- the antibody may also be produced by a combination of recombinant DNA techniques with other techniques known in the art.
- Presently preferred labels for the antibody molecules are radioopaque labels, i.e. heavy metals, or enzymatic markers, known in the art.
- a serum-based method for detecting a tumor in a subject comprises isolating serum from the subject, contacting the serum with a first detectable marker which binds specifically to at least a portion of a polypeptide of this invention and detecting the marker so bound.
- the presence of bound marker indicates the presence of a tumor.
- the first detectable marker is preferably a labelled antibody which may be free, i.e. in a solution, or may be bound to a matrix, i.e. a matrix such as polystyrene beads or the wall of a tube.
- Radiolabels i.e. 35S, heavy metals, or enzymatic labels.
- the bound antibodies will be detectable by autoradiography, scintillation counting or by colorimetry or by various other means.
- the second detectable marker of this invention will also be labelled antibody molecule and may specifically bind to the polypeptide or to the first marker or to a combination.
- the second marker will be labelled by radiolabels, heavy metals or enzymes, and detectable by means similar to those used to detect the first marker.
- the second marker may also be detectable by eye if it causes precipitation of the first marker-polypeptide complex.
- a method for treating in a subject a tumor induced by an activated rosl oncogene comprises isolating an immunoglobulin molecule which specifically binds to at least a portion of a polypep tide encoded by the activated rosl oncogene, attaching to the immunoglobulin molecule so isolated a substance which substantially limits the growth of a tumor or which destroys tumors to produce an antitumor immunoglobulin molecule, and contacting the tumor with an effective amount of the antitumor immunoglobulin molecule so produced, thereby limiting tumor growth or destroying the tumor.
- tumors in a subject which may be treated according to the method of the present invention are those arising from astrocytoma and glioblastoma cells.
- the immunoglobulin molecule is preferably obtained by contacting the immune system of an animal with at least a portion of a polypeptide encoded by the associated rosl oncogene, or with a synthetic amino acid sequence substantially similar to a portion of the polypeptide, to produce a specific antibody molecule, and isolating the antibody therefrom.
- the immunoglobulin molecule may also be produ-ced by a combination of recombinant DNA techniques and other techniques known in the art.
- Substances which substantially limit the growth of a tumor or which destroy tumors are known in the art, and may be molecules such as interferon, tumor necrosis factors, or radioactively labelled amino acids.
- NIG3T3 cells at 8x10 cells/plate were cotransformed with 300 ng of pKOneo plasmid DNA (32) and 5 micrograms of each of the cosmid fragments.
- the selection for G418 antibiotic resistance and the tumorigenicity assay were performed as previously described (8).
- Cell cultures were established from excised tumors following surgical removal and mincing, and were maintained in Dulbecco's medium plus 10% calf serum under our standard culture conditions. Nomenclature for tumors and cell lines derived from them is as follows. Independent secondary tumors and cell lines derived from MCF- 7-3 DNA are called MCF-7-3n where n is a number. Independent tertiary tumors and cell lines derived from MCF-7-3n DNA are called MCF-7-3n-m where m is a number.
- DNAs were prepared from tumors or human placentas as previously described (8). Genomic libraries were constructed from placental or MCF-7-3-4 tumor DNAs by partial cleavage with EcoRI and cloning into the cosmid vector pHC79 (10). Appropriate fragments from a previously isolated lambda library (8) were used. for colony filter hybridization. Additional overlapping cosmid clones were then isolated by hybridization with appropriate probes isolated from cosmid clones. cDNAs were synthesized from polyA mRNA- isolated from the nude mouse tumor derived cell line MCF-7-3-7 (13). The cDNA library was constructed in lambda gtlO (11). Phages containing mcf3 cDNAs were isolated by plaque hybridization, initially with the EcoRI fragment 1.4 kbp in length isolated from cosmid clone 101, and later with fragments isolated from cDNA clones.
- DNA and DNA analysis Southern blot analyses under high and low stringency conditions were performed as previously described (25,19).
- DNA sequences were determined by the dideoxy method of Sanger et al. (21) as modified by Biggin et al. (2). Both strands of the coding sequences of the mcf3 cDNA were sequenced. To localize the exon sequences. Southern analysis was performed using cDNA fragments as probes. A synthetic oligonucleotide of the sequence 5'-CCAACTATAATAGTAAGTATG-3', which corresponds to the noncoding strand of the sequences encoding amino acids 10 to 15, was used as probe to localize the exon encoding the transmembrane domain.
- This oligonucleotide hybridized to the EcoRI fragment 4.8 kbp in length, and was used as primer for sequencing double stranded DNA fragments from both placental and mcf3 cosmid clones, by a modification of the dideoxy method (6).
- the oligonucleotides used in this study were synthesized on an Applied Biosystems DNA Synthesizer and purified from polyacrylamide gels as described (35).
- a cosmid library was constructed from human placental DNA. Appropriate DNA fragments from the mcf3 locus were used for screening this library, and five independent cosmid clones were obtained. The DNAs present in these clones could not be aligned over their full length with the mcf3 locus. Additional cloning and Southern analysis indicated that the different structure of the placental cosmid clones and the mcf3 locus was not an artifact of cosmid cloning.
- the mcf3 locus was created by DNA rearrangement involving the fusion of at least three separate fragments of human DNA.
- the first of these is a fragment of human DNA located at what we later show to be the 5'-end of the mcf3 oncogene and it extends from the left end of the map of the mcf3 locus to the EcoRI fragment 3.7 kbp in length. This fragment is connected by DNA of unknown origin to another fragment of the human genome which contains the majority of the coding sequences of mcf3.
- the second piece of human DNA extends from the EcoRI fragment 4.8 kbp in length to the right end of the map of the mcf3 locus.
- the DNA rearrangements which created the mcf3 locus are probably of functional significance since they span the regions that were required in the cotransfection studies described above.
- RNA from a cell line established from the- secondary nude mouse tumor MCF-7-3-7 was analysed.
- Various fragments from the mcf3 locus were used as probes in Northern blots.
- the 1.4 kbp EcoRI fragment contained in cosmid 101 detected several RNA species of 2.8 kbp to 3.3 kbp length in RNA from MCF-7-3-7 cells. These RNA species were not found in RNA from normal NIH3T3 cells.
- the EcoRI fragment 1.4 kbp in length was then used as probe for screening a cDNA library.
- the library was constructed by cloning of cDNAs synthesized from polyA+ RNA of the MCF-7-3-7 cell line into the lambda gtlO cloning vector (11).
- the 3'-end of one transcript was localized in cDNA clone M3.9, which contained a polyA+ tail. This also determined the direction of transcription in the mcf3 locus which was subsequently verified by SI mapping.
- v-ros probe hybridized most strongly with an EcoRI fragment in human DNA 2.9 kbp in length, corresponding to the same fragment of the mcf3 locus. It was concluded therefore that a major portion of the mcf3 coding sequence is derived from the gene in huraans most closely related to the v-ros gene. We call this gene rosl, since other human genes related to the v-ros or the mcf3 gene may exist.
- v-ros Five of the oncogenes known to encode oncogenic tyrosine kinases, v-ros (16), v-erbB (33), v-fms (9), neu (1) and trk (14), have hydrophobic potential membrane spanning domains.
- the membrane spanning domains of these proteins are always encoded 5'-to sequences encoding the kinase domain. Inspection of the mcf3 nucleotide sequence shows that it can encode a highly hydrophobic stretch of 21 amino acids immediately followed by a stretch rich in positively charge amino acids (see Figure 1, boxed sequence). These features are commonly found in membrane spanning domains.
- the hydrophobic sequences of v-ros are 30 amino acids in length, longer than in mcf3 and longer than is needed for such a domain.
- the portion of the mcf3 cDNA encoding the potential transmembrane domain derived from the human rosl gene or from other parts of the mcf3 locus was also studied.
- restriction endonuclease analysis Southern blotting and hybridization with synthetic oligonucleotides, two coding exons were localized and sequenced within the 4.8 kbp EcoRI fragment which contains the breakpoint in the rosl derived part of the mcf3 locus.
- the positions of the deduced splice junctions are included in Figure 1.
- One exon encodes sequences for the putative intracellular domain and for one amino acid of the potential transmembrane domain.
- the other encodes sequences from the potential transmembrane domain, and for eight amino acids of the putative extracellular domain. This last exon is found also in placental clones containing the human rosl gene and therefore does not derive from rearranged sequences. Horaologs to the eight amino . acids of the putative extracellular domain are not found in the avian v-ros gene.
- the mcf3 gene derives from a rearrangement involving the human rosl gene, and putafcive extracellular sequences of this gene have been lost. Since similar rearrangements have occurred during the biogenesis of the v-ros (16), v-erbB (33) and or trk (14) genes, they are probably of functional significance. It was thus of interest to determine whether DNA from the MCF-7 cell line used in the original cotransformation studies contained the rearranged mcf3 locus, or whether this rearrangement was introduced during DNA transfer. The structure of the locus in the MCF-7 cell line was analyzed by Southern blotting and compared to either the normal configuration of the locus in placenta or the rearranged configuration in MCF-7-3 and its derived tumors.
- the probe used for these experiments contained sequences from the 4.8 kb EcoRI fragment localized in the middle of the mcf3 locus which contains one of the breakpoints introduced during rearrangement.
- This probe hybridized to a BamHI fragment approximately 12 kb in length in tumor DNAs containing the rearranged mcf3 locus but not to a band approximately 10 kb in length in DNA from MCF-7 cells and from placenta. Therefore, the rearrangement responsible for the creation of the mcf3 locus seems not to have occurred in MCF-7 DNA. The alteration is only found in DNA from MCF-7-3 and its derived tumors and must therefore have occurred during or after DNA transfer into
- RNA from the MCF-7 cell line was analyzed by Northern blotting and by RNA protection studies (see Materials and Methods). Expression of the rosl gene in MCF-7 cells was not detected. If expressed at all, levels of the rosl transcript in MCF-7 cells must be fifty-fold lower than levels found in cells transformed with the mcf3 locus.
- the oncogenic potential of the human rosl gene may have been activated by rearrangement and gene amplification occurri ⁇ ag during or after gene transfer.
- v-erbB 33
- v-fms a
- neu (1) trk
- v-ros and cellular ros genes (16,17).
- the cellular analogs of the v-erbB and the v-fms genes probably encoded the receptors for the epidermal growth factor and macrophage colony-stimulating factor, respectively.
- the cellular ros gene very likely encodes a hormone receptor as well.
- the malignant forms include the highly malignant glioblastoma multiforme. Interestingly, a good correlation between rosl expression and the degree of malignancy was found. This raises the possibility that the expression of rosl contributes to the malignant phenotype of this particular cell type, and may be helpful in the classification and diagnosis of this type of cancer.
- mcf3 an oncogene called mcf3 (8).
- the gene was detected by a combination of DNA mediated gene transfer and a tumorigenicity assay in nude mice.
- the DNA originally used in the first transfections came from MCF-7 cells, a human mammary carcinoma cell line (8).
- the mcf3 gene was isolated by molecular cloning and its structure was compared to the structure of its normal counterpart in human placental DNA.
- the mcf3 oncogene was a product of a major DNA rearrangement. DNA cotransfection studies indicated that this rearrangement spanned functionally important domains of mcf3.
- tumorigenicity assay may be unreliable for the detection of oncogenes in tumor DNAs, it may be a good method for searching for protooncogenes which can be activated by rearrangement or amplification.
- rosl DNA fragment in human DNA, and the gene from which mcf3 was derived was designated as rosl. This nomenclature is used since there may be other ros-related genes in the human genome. In the human genome, the rosl gene is localized on chromosome 6, bands q11-q22 (20). Translocations of chromosome 6 in this region have been previously observed in human tumors. Thus, although rosl is probably not activated in MCF-7 tumor cells, it is possible that chromosomal alterations affect the rosl locus and lead to an activation of its oncogenic potential in other tumor cells.
- the cellular ros gene very likely encodes a hormone receptor as well.
- the tyrosine kinase most closely related to ros is the insulin receptor (29).
- a stretch of very high (75%) homology to the insulin receptor exists between position 245 and 288 in rosl, which can be aligned with positions 178 to 215 in the cAMP dependent protein kinase (cAPK). Since cys 198 of the cAPK is protected from chemical modification by peptide substrates, this region has been implicated in substrate binding (5).
- the high degree of homology between the amino acid sequences of rosl and the insulin receptor in this putative substrate binding domain might indicate a similar substrate specificity for the tyrosine kinase activities of these two proteins.
- Insertion of the provirus into the middle of the c-erbB gene leads to the production of a truncated erbB transcript which encodes only 64 amino acids of the extracellular domain but an intact membrane spanning and intracellular domain.
- trk seems to have been formed by a somatic rearrangement that replaced the extracellular domain of a putative transmembrane receptor with the first 221 amino acids of a nonmuscle tropomyosin protein (14).
- the nature of the sequences in mcf3 which have replaced the extracellular domain of the rosl gene has not been analyzed. However, one should be cautious in concluding that the loss of these extracellular sequences has led to the activation of rosl.
- the amino acid sequence of mcf3 from position 1 to 392 is identical to the deduced coding sequence of rosl as determined from human placental DNA, but the corresponding coding sequences C-terminal to amino acid 392 have not yet been determined from rosl. Although gross structural differences between the DNA of mcf3 and ros1 coding for the C-terminal part of the protein could not be detected, any subtle changes like point mutations or small deletions would not have been detected. Moreover, the rearranged rosl gene is very highly amplified in all mcf3 transformed NIH3T3 cells we have examined. It is not yet possible to assess the relative contributions of rearrangement and amplification of this gene on its oncogenicity.
- Proto-oncogene c-ros codes for a molecule with structural features common to those of growth factor receptors and displays tissue-specific and developmentally regulated expression. Mol. Cell. Biol. 6:1478-1486.
- the c-fms proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor, CSF-1. Cell 41:665-676.
- the erbB gene of avian erythroblastosis virus is a member of the src gene family. Cell 35:71-78.
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Abstract
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
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US86556986A | 1986-05-21 | 1986-05-21 | |
US865569 | 1986-05-21 | ||
US4891587A | 1987-05-19 | 1987-05-19 | |
US48915 | 1987-05-19 |
Publications (2)
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EP0267275A1 true EP0267275A1 (fr) | 1988-05-18 |
EP0267275A4 EP0267275A4 (fr) | 1989-09-19 |
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EP19870903664 Withdrawn EP0267275A4 (fr) | 1986-05-21 | 1987-05-21 | SEQUENCE ADN CODANT L'ONCOGENE -i(ROS), POLYPEPTIDES CODES A PARTIR DE CELLE-CI, ET DIAGNOSTIC ET AUTRES PROCEDES BASES SUR CETTE SEQUENCE. |
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EP (1) | EP0267275A4 (fr) |
JP (1) | JPH01500481A (fr) |
WO (1) | WO1987007305A1 (fr) |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
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US5085983A (en) * | 1988-08-19 | 1992-02-04 | City Of Hope | Detection of human tumor progression and drug resistance |
AU631788B2 (en) * | 1988-04-15 | 1992-12-10 | Montefiore Medical Center | Method for determining state of malignant and pre-malignant progression |
WO1990008160A1 (fr) * | 1989-01-20 | 1990-07-26 | Imperial Cancer Research Technology Ltd. | Peptides analogues a des recepteurs de facteur de croissance ne presentant pas d'activite de tyrosine kinase |
CA2040099A1 (fr) * | 1990-05-01 | 1991-11-02 | Mariano Barbacid | Tyrosine-kinase-negative trkb |
US6696548B2 (en) * | 1993-12-03 | 2004-02-24 | St. Jude Children's Research Hospital | Antibodies for recognition of alk protein tyrosine/kinase receptor |
EP2333103A1 (fr) | 2009-12-11 | 2011-06-15 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Diagnostic différentiel et thérapie pour les inhibiteurs de la kinase |
EP2336171A1 (fr) * | 2009-12-11 | 2011-06-22 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Nouvelles cibles pour le traitement des maladies proliférantes |
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EP0119702A2 (fr) * | 1983-02-14 | 1984-09-26 | Arup Sen | Polypeptides synthétiques obtenus à partir d'oncogènes viraux |
-
1987
- 1987-05-21 WO PCT/US1987/001215 patent/WO1987007305A1/fr not_active Application Discontinuation
- 1987-05-21 EP EP19870903664 patent/EP0267275A4/fr not_active Withdrawn
- 1987-05-21 JP JP62503312A patent/JPH01500481A/ja active Pending
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EP0119702A2 (fr) * | 1983-02-14 | 1984-09-26 | Arup Sen | Polypeptides synthétiques obtenus à partir d'oncogènes viraux |
Non-Patent Citations (8)
Title |
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CHEMICAL ABSTRACTS, vol. 101, no. 23, 12th March 1984, page 141, abstract no. 205162e, Columbus, Ohio, US; O. FASANO et al.: "New human transforming genes detected by a tumorigenicity assay", & MOL. CELL. BIOL. 1984, 4(9), 1695-705 * |
CHEMICAL ABSTRACTS, vol. 104, no. 9, 3rd March 1986, page 184, abstract no. 63206h, Columbus, Ohio, US; J.J.M. VAN DONGEN et al.: "Immunoglobulin chain gene expression in Burkitt lymphoma derived cell lines", & UCLA SYMP. MOL. CELL. BIOL., NEW SER. 1985, 28 (LEUK.: RECENT ADV. BIOL. TREAT.) 481-9 * |
CHEMICAL ABSTRACTS, vol. 96, no. 23, 7th June 1982, pages 182-183, abstract no. 194360t, Columbus, Ohio, US; M. SHIBUYA et al.: "Cellular sequences related to three new onc genes of avian sarcoma virus (fps, yes, and ros) and their expression in normal and transformed cells", & J. VIROL. 1982, 42(1), 143-52 * |
JOURNAL OF VIROLOGY, vol. 53, no. 3, March 1985, pages 879-884, American Society for Microbiology; W.S. NECKAMEYER et al.: "Nucleotide sequence of avian sarcoma virus UR2 and comparison of its transforming gene with other members of the tyrosine protein kinase oncogene family" * |
MOLECULAR & CELLULAR BIOLOGY, vol. 6, no. 9, September 1986, pages 3109-3116, American Society for Microbiology; C. BIRCHMEIER et al.: "Characterization of an activated human ros gene" * |
MOLECULAR AND CELLULAR BIOLOGY, vol. 6, no. 5, May 1986, pages 1478-1486, American Society of Microbiology; W.S. NECKAMEYER et al.: "Proto-oncogene c-ros codes for a molecule with structural features common to those of growth factor receptors and displays tissue-specific and developmentally regulated expression" * |
PROCEEDINGS NATIONAL ACADEMY OF SCIENCES USA, vol. 83, no. 5, September 1986, pages 6568-6572; L. NAGARAJAN et al.: "The human c-ros gene (ROS) is located at chromosome region 6q16-6q22" * |
See also references of WO8707305A1 * |
Also Published As
Publication number | Publication date |
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EP0267275A4 (fr) | 1989-09-19 |
WO1987007305A1 (fr) | 1987-12-03 |
JPH01500481A (ja) | 1989-02-23 |
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