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WO1999060116A1 - Acide nucleique a regulation negative de la metastase (drim) codant pour une proteine, et utilisation diagnostique et therapeutique de cet acide nucleique - Google Patents

Acide nucleique a regulation negative de la metastase (drim) codant pour une proteine, et utilisation diagnostique et therapeutique de cet acide nucleique Download PDF

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WO1999060116A1
WO1999060116A1 PCT/EP1999/003396 EP9903396W WO9960116A1 WO 1999060116 A1 WO1999060116 A1 WO 1999060116A1 EP 9903396 W EP9903396 W EP 9903396W WO 9960116 A1 WO9960116 A1 WO 9960116A1
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dna
nucleic acid
polypeptide
drim
gene
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Ulrich Weidle
David Tarin
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Roche Diagnostics GmbH
Oxford University Innovation Ltd
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Roche Diagnostics GmbH
Oxford University Innovation Ltd
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Priority to AU42635/99A priority Critical patent/AU4263599A/en
Publication of WO1999060116A1 publication Critical patent/WO1999060116A1/fr
Anticipated expiration legal-status Critical
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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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention relates to a new nucleic acid, down-regulated in metastasis (DRIM) and to the corresponding protein produced by recombinant techniques as well as nucleic acids which code for proteins with DRIM activity, methods of use and of production.
  • DRIM down-regulated in metastasis
  • the invention comprises especially novel therapeutic compositions comprising recombinant proteins produced using nucleic acid sequences encoding proteins with DRIM activity.
  • the invention is based on a new protein with antimetastatic activity, which
  • a) is coded by the DNA sequence shown in SEQ ID NO: 1
  • b) is coded by DNA sequences which hybridize under stringent conditions with the complementary sequence of the DNA sequence shown in SEQ ID NO:l.
  • the protein can be defined by its DNA sequence and by the amino acid sequence derived therefrom.
  • the DRIM protein can occur in natural allelic variations which differ from individual to individual. Such variations of the amino acids are usually amino acid substitutions. However, they may also be deletions, insertions or additions of amino acids to the total sequence.
  • the DRIM protein according to the invention - depending, both in respect of the extent and type, on the cell and cell type in which it is expressed - can be in glycosylated or non-glycosylated form the DRIM protein according to SEQ ID NO:2 is preferred.
  • DRIM mRNA is significantly more strongly expressed in non-metastasizing breast cancer cells.
  • hybridize under stringent conditions means that two nucleic acid fragments are capable of hybridization to one another under standard hybridization conditions described in Sambrook et al., "Expression of cloned genes in E. coli” in Molecular Cloning: A laboratory manual (1989) Cold Spring Harbor Laboratory Press, New York, USA, 9.47 - 9.62 and 11.45 - 11.61.
  • stringent conditions refer to hybridization in 1 mol/1 NaCl, 1 % SDS and 10% dextransulfate. This is followed by two washes of the filter at room temperature of 5 minutes in 2 x SSC and one final wash for 30 minutes. This final wash may be at 0.5 x SSC, 0.1 % SDS, more preferably at 0.2 x SSC, 0.1 % SDS and most preferably at 0.1 x SSC, 0.1 % SDS, final wash taking place at 65°C.
  • Those of ordinary skilled in the art will recognize that other conditions will afford the same degrees of stringency and are understood as "under stringent conditions”.
  • DRIM is a protein which is active in its glycosylated or unglycosylated form.
  • the unglycosylated form can be produced by recombinant technology in prokaryotic cells.
  • DRIM activity means an antimetastatic activity. Such activity can be achieved by transfecting an expression vector for DRIM into a metastasizing cell line (e.g., as described by Caillou (1978)), establishment of stable transfectants and evaluation of their metastatic properties by inoculation into nude mice.
  • a metastasizing cell line e.g., as described by Caillou (1978)
  • Proteins with DRIM activity means also proteins with minor amino acid variations but with substantially the same activity. Substantially the same means that the activities are of the same biological properties and preferably at least 85% homology in amino acid sequence. More preferably, the amino acid sequences are at least 90% identical.
  • DRIM can be purified from non-metastasizing breast cancer cells by affinity chromatography using a monoclonal antibody against DRIM. It is also preferred to use other known protein purification techniques, including immunoprecipitation, gel filtration, ion exchange, chromatography, chromatofocussing, isoelectric focussing, selective precipitation, electrophoresis, and the like. Fraction isolated during purification procedures can be analyzed for the presence of DRIM activity by using DRIM specific antibodies.
  • the protein according to the invention can also be produced by recombinant means.
  • Non-glycosylated DRIM protein is obtained when it is produced recombinantly in prokaryotes.
  • nucleic acid sequences provided by the invention it is possible to search for the DRIM gene or its variants in genomes of any desired cells (e.g. apart from human cells, also in cells of other mammals), to identify these and to isolate the desired gene coding for the DRIM protein.
  • Such processes and suitable hybridization conditions are known to a person skilled in the art and are described, for example, by Sambrook, J., et al., "Expression of cloned genes in E.
  • DRIM protein derivatives can, for example, be modified in individual or several amino acids by substitution, deletion or addition.
  • the derivatization can, for example, be carried out by means of site directed mutagenesis. Such variations can be easily carried out by a person skilled in the art (J. Sambrook, B.D. Hames, loc. cit.). It merely has to be ensured that the characteristic properties of the DRIM protein (inhibition of the aforementioned cell lines) are preserved.
  • the invention therefore in addition concerns a DRIM protein which is a product of a prokaryotic or eukaryotic expression of an exogenous DNA.
  • the invention further concerns a nucleic acid molecule for use in securing expression in a prokaryotic or eukaryotic host cell of a DRIM protein and is selected from the group comprising
  • nucleic acid sequences which hybridize under stringent conditions with one of the sequences from a), and c) nucleic acid sequences which, if there was no degeneracy of the genetic code, would hybridize with one of the sequences stated in a) or b).
  • the protein according to the invention can be obtained in a reproducible manner and in large amounts.
  • the nucleic acid is integrated into suitable expression vectors, according to methods familiar to a person skilled in the art.
  • suitable expression vectors preferably contains a regulatable/inducible promoter. These recombinant vectors are then introduced for the expression into suitable host cells such as, e.g., E.
  • coli as a prokaryotic host cell or Saccharomyces cerevisiae, teratocarcinoma cell line PA-1 sc 9117 (B ⁇ ttner et al., Mol. Cell. Biol. 11 (1991) 3573-3583), insect cells, CHO or COS cells as eukaryotic host cells and the transformed or transduced host cells are cultured under conditions which allow expression of the heterologous gene.
  • the isolation of the protein can be carried out according to known methods from the host cell or from the culture supernatant of the host cell. Such methods are described for example by Ausubel I., Frederick M., Current Protocols in Mol. Biol. (1992), John Wiley and Sons, New York. Also in vitro reactivation of the protein may be necessary.
  • the detection of transformed or transduced host cells which recombinantly produce the DRIM protein and the purification of the protein are preferably carried out by means of antibodies which bind to this protein.
  • Such antibodies can be obtained in a simple manner according to known methods by using the protein according to the invention as an antigen or an immunogen.
  • the invention therefore in addition concerns the use of the protein with DRIM activity according to the invention for the production of antibodies which bind to this protein.
  • Anti-DRIM antibodies are produced by immunization of appropriate vertebrate hosts with purified DRIM or polypeptide derivatives of DRIM, preferably with an adjuvant. Said techniques are well-known in the literature and are described, for example, by Harlow and Lane eds., Antibodies: A laboratory manual (1988), Cold
  • animals which are usually used for this purpose such as in particular, sheep, rabbits or mice, are immunized with the protein according to the invention and subsequently the antiserum is isolated from the immunized animals according to known methods or spleen cells of the immunized animals are fused with immortalized cells, such as e.g. myeloma cells, according to the method of K ⁇ hler and Milstein (Nature 256 (1975) 495-497).
  • Those cells which produce a monoclonal antibody against the DRIM protein are selected from the hybridoma cells obtained in this way and cloned.
  • the monoclonal or polyclonal antibodies obtained in this way can be bound to a support material, such as e.g. cellulose, for an immunoabsorptive purification of the melanoma-inhibiting protein.
  • antibodies of this kind can be used for the detection of the DRIM protein in samples, such as e.g. cut tissue or body fluids.
  • the invention therefore additionally concerns antibodies against the DRIM protein which are obtainable by immunizing an animal with a DRIM protein and isolating the antibodies from the serum or spleen cells of the immunized animals.
  • the invention in addition concerns the use of a protein according to the invention for the production of a therapeutic agent which can be used in tumor therapy.
  • the protein according to the invention is processed, if desired together with the usually used auxiliary agents, fillers and/or additives, in a pharmaceutical formulation for the said therapeutic applications.
  • the invention therefore in addition concerns a therapeutic composition containing a DRIM protein according to the invention and if desired together with the auxiliary agents, fillers and/or additives that are usually used.
  • the invention further concerns the use of sequences of the DRIM gene, preferably nucleic acid molecules coding for a protein having DRIM activity, or activating polynucleotides from the 5' untranslated region, in gene therapy, and in particular, for the production of medicaments for gene therapy.
  • DRIM locally or in distant metastases
  • Gene therapy of somatic cells can be accomplished by using, e.g., retroviral vectors, other viral vectors, or by non-viral gene transfer (for clarity cf. T. Friedmann, Science 244 (1989) 1275; Morgan 1993, RAC DATA MANAGEMENT REPORT, June 1993).
  • Vector systems suitable for gene therapy are, for instance, retroviruses (Mulligan,
  • Epstein Barr virus (Margolskee et al, Mol. Cell. Biol. 8 (1988) 2937) or herpes simplex virus.
  • nucleic acid preferably DNA
  • nucleic acid usually “nude” nucleic acid, preferably DNA, is used, or nucleic acid together with an auxilary such as, e.g., transfer reagents (liposomes, dendromers, polylysine-transferrin-conjugates
  • Another preferred method of gene therapy is based on homologous recombination.
  • either the gene coding for the DRIM protein can be inserted in one or more copies into the genome of somatic cells and/or the DRIM gene endogenously present in the cells can be modulated, preferably activated.
  • the targeting DNA is a DNA which is complementary (homologous) to a region (preferably within or proximal to the DRIM gene) of the genomic DNA.
  • a region preferably within or proximal to the DRIM gene
  • the targeting DNA and the genomic DNA are in close proximity to one another they will hybridize and form a double-stranded helix.
  • the DRIM gene fragment and the targeting DNA can be integrated into the genome by means of occurrence of recombination. This homologous recombination can be carried out both in vitro and in vivo (in the patient), in allogeneic or autologous cells.
  • a DNA which codes for a protein having DRIM activity a fragment which inhibits DRIM expression (knock-out sequence) or a fragment capable of activating, after integration into the genome of a cell, expression, in this cell, of a protein having DRIM activity.
  • a fragment may be, for example, a promoter and/or enhancer region which is heterologous to the corresponding DRIM region or which, after integration into the DRIM gene, activates the actually silent or to a little extent expressed DRIM gene transcriptionally and/or translationally.
  • one or more DRIM genes are newly introduced into the target cell, or the essentially transcriptionally silent gene in the genome of a mammalian cell is activated in such fashion that the mammalian cell is enabled to produce endogenous DRIM protein.
  • a DNA construct is inserted into the genome by homologous recombination, the DNA construct comprising the following: a DNA regulatory element capable of stimulating expression of this gene if operatively linked thereto; and one or more DNA target segments which are homologous to a region in this genome, which region is within or proximal to this gene.
  • This construct is inserted into the genome of the mammalian cell in such fashion that the regulatory segment is operatively linked to the gene which codes for the protein having DRIM activity.
  • the construct further comprises amplifying sequences, especially if genes coding for proteins with DRIM activity are inserted into the cell.
  • the construct For the introduction of DRIM genes into the target cells, the construct comprises a regulatory element, one or more DRIM genes and one or more target segments.
  • the target segments are chosen in such a way that they hybridize with an appropriate region of the genome, whereby, after homologous recombination, the inserted exogenous DRIM genes are expressed.
  • homologous recombination takes place during DNA replication or mitosis of the cells.
  • a DNA of this kind can be used for the production of an agent for therapeutic treatment of tumors or for the production of homologous or heterologous DRIM protein in a host organism.
  • test on the basis of the nucleic acid sequences of the DRIM protein provided by the invention which can be used to detect nucleic acids which code for DRIM proteins.
  • a test can for example be carried out in cells or cell lysates and by means of nucleic acid diagnostics.
  • the sample to be examined is brought into contact with a probe which would hybridize with the nucleic acid sequence coding for the DRIM protein.
  • a hybridization between the probe and nucleic acids from the sample indicates the presence of expressed DRIM proteins.
  • nucleic acid of the sample which codes for a DRIM protein is amplified before testing, e.g. by the well-known PCR technique.
  • a derivatized (labelled) nucleic acid probe is usually used in the field of nucleic acid diagnostics.
  • This probe is brought into contact with a carrier-bound denatured DNA or RNA from the sample and in this process the temperature, ionic strength, pH value and other buffer conditions are selected in such a way that - depending on the length of the nucleic acid sample and the resulting melting temperature of the expected hybrid - the labelled DNA or RNA can bind to homologous DNA or RNA (hybridization, see also Southern,
  • Suitable carriers are membranes or carrier materials based on nitrocellulose (e.g. Schleicher and Sch ⁇ ll, BA 85, Amersham Hybond, C), reinforced or bound nitrocellulose in a powder form or nylon membranes derivatized with various functional groups (e.g. nitro group) (e.g. Schleicher and
  • the hybridized DNA or RNA is then detected by incubating the carrier, after thorough washing and saturation to prevent unspecific binding, with an antibody or antibody fragment.
  • the antibody or antibody fragment is directed towards the substance incorporated into the nucleic acid probe during the derivatization.
  • the antibody is in turn labelled. It is, however, also possible to use a directly labelled DNA. After incubation with the antibodies, it is washed again in order to only detect specifically bound antibody conjugates. The determination is then carried out via the label of the antibody or antibody fragment according to well-known methods.
  • the detection of DRIM expression can be carried out for example as:
  • serum analysis e.g. cell type analysis of cells in serum by slot-blot analysis
  • the nucleic acid probe is incubated with the nucleic acid from the sample and the hybridization of the nucleic acid in the sample and nucleic acid probe is detected, if desired, via a further binding partner.
  • DRIM is a valuable prognostic marker in tumor diagnostics (metastasis and tumor progression).
  • SEQ ID NO 1 denotes the nucleotide sequence of the DRIM cDNA and polypeptide sequence.
  • SEQ ID NO 2 denotes the polypeptide sequence of DRIM. Description of the Figures
  • Fig. 1 shows the expression of DRIM messenger RNA in selected tissues.
  • Clontech filters with immobilized polyA + RNA were hybridized with an ⁇ P-labeled probe covering part of the 3 'untranslated region of DRIM cDNA.
  • Lanes a, heart; b, brain; c, placenta; d, lung; e, liver; f, skeletal muscle; g, kidney; h, pankreas; i, spleen; j, thymus; k, prostate; 1, testis; m, ovary; n, small intestine; o, colon; p, appendix; q, peripheral blood lymphocytes; r, bone marrow; s, fetal liver.
  • Fig ' 2 shows the expression of DRIM messenger RNA in selected tumor cell lines.
  • Clontech filters with immobilized polyA + RNA were hybridized with ⁇ 32p-labeled probe covering part of the 3 'untranslated region of DRIM cDNA.
  • Lanes a, HL60 cells; b, HeLa cells; c, K562 cells; d, MOLT-4 cells; e, Raji cells; f, SW480 cells (colorectal carcinoma), lung carcinoma cell line A-549; g, lung carcinoma A549; and h, G361 melanoma cells.
  • Fig. 3 shows a Northern blot analysis of DRIM messenger RNA expression in selected mammary carcinoma cell lines.
  • PolyA + RNA was extracted from confluent cell lines, separated on a 1 % agarose gel, transferred to a nitro-cellulose membrane and hybridized to a P-labeled probe derived from the 3 'untranslated region of DRIM.
  • MCF-7 ADR ; j, LCC-1; k, LCC-2; 1, LLC-9.
  • Fig. 4 shows differential expression of DRIM messenger RNA in cell lines 4A4 and 2C5 displayed by Northern blot analysis.
  • RNA derived from different cell culturing experiments (lanes a and b versus lanes c and d) is displayed.
  • PolyA+RNA from cell lines 2C5 (lanes a and c) and 4A4 (lanes b and d) was electrophoresed on a 1% agarose gel, transferred to a nitrocellulose membrane and hybridized to an G ⁇ 32_P-labeled fragment covering part of the 3' untranslated region of DRIM cDNA.
  • the blot was rehybridized to a human ⁇ -actin probe as an internal reference. Size of markers is indicated.
  • Athymic mice (MFlNa) were obtained from the animal breeding facility at the John Radcliffe Hospital, Oxford University, UK. Mice were used at 6 - 8 weeks of age and were kept in filter-top boxes in a nude mouse isolation suite for the duration of the experiments.
  • A4A and 2C5 cells are cloned sublines of MDA-MD-435, a mammary carcinoma cell line described by Caillou, R., et al. (In vitro 14 (1978) 911-915), which we isolated by the limiting dilution technique. These cells were maintained in Dulbeccos's Modified Eagle Medium (DMEM) (Flow Laboratories, Irvine, U.K.) supplemented with 5 % new born calf serum, sodium pyruvate, L-glutamine
  • DMEM Dulbeccos's Modified Eagle Medium
  • the cell line 2C5 was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg lb, D-38124 Braunschweig on May 5, 1998 and has been accorded the Deposition No. DSM ACC 2351.
  • MDA-MB435 From MDA-MB435, first MDA-MB-lung was established from pooled lung metastases arising in a nude mouse which had been injected with MDA-MB 435. MDA-BM-lung is much more metastatic than the parent line. A series of clones were obtained by serial dilution of MDA-MB-lung in 96 well plates and subsequent clonal expansion. Clone 4A4 is metastatic, whilst clone 2C5 is not metastatic.
  • Tumorigenicity and metastasis formation in vivo The tumorigenicity and spontaneous metastatic capability of the cells were determined by injection of groups of nude mice with 1 x 10 6 cells in 0.1 ml DMEM into the lower right hind flank, either subcutaneously or in the right posterior mammary fat pad. The animals were monitored every 2 - 3 days for up to 5 months for state of health and tumor growth. The rate of primary tumor growth of two of the MDA clones (4A4 and 2C5) was determined by plotting the means of two orthogonal diameters of the tumors, measured at 20 day intervals up to 100 days after injection. All animals were killed and autopsied 5 months after inoculation unless moribund earlier.
  • the experimental metastatic potential of the cell lines was assessed by intravenous injection of 1 x 10 5 in 0.1 ml serum-free DMEM into the lateral tail vein of nude mice. Recipient animals were killed and autopsied 2 months after injections.
  • Metastasis formation was assessed by macroscopic observation of all major organs for secondary tumors and confirmed by histological examination of organs and lymph nodes. Tissue samples for histological analysis were fixed in 10 % neutral formalin and embedded in paraffin wax for sectioning and staining.
  • DD-PCR Differential Display Polymerase chain reaction
  • RNA Isolation System Promega Corp., Madison, WI. Elimination of contaminating traces of DNA from total RNA samples was performed by digestion at 38°C for 30 minutes with RNase-free DNasel using the MessageClean kit (GenHunter Corp., Brookline, US).
  • DNA-free total RNA (0,2 ⁇ g from 4A4 and 2C5 cells) was used as a template for first strand synthesis in the presence of 10 ⁇ M T 12 MG, T ⁇ 2 MC, T 12 MA and T 12 MT anchored primers (where M is threefold degenerate for G, A and C), 1 x reverse transcriptase buffer [125 mM Tris-Cl, pH 8.3, 188 mM KC1, 7.5 mM MgCl 2 , 25 mM dithiothreitol (DDT)] and 250 ⁇ M dNTP mix. The solution was heated to 65°C for 5 minutes and cooled to 37°C for 10 minutes, and then 200 units of
  • MMLV Moloney murine leukemia virus reverse transcriptase
  • the PCR was performed in a mix containing 0.1 volume of reverse transcription reaction mixture, 10 ⁇ M of the respective T 12 MN anchored primer, 2 ⁇ M arbitrary
  • PCR included a total of 40 cycles at 94°C for 30 seconds, 40°C for 2 minutes, 72°C for 30 seconds, and finally 5 minutes at 72°C.
  • the PCR products were heated at 80°C for 2 minutes and then loaded on a denaturing 5% polyacrylamide sequencing gel for electrophoresis.
  • the dried gel was exposed to Kodak BioMax MR film for 48 hours at room temperature and the autoradiogram was analyzed with respect to differentially expressed genes.
  • the reaction displaying unique fragments in one of the two cell lines was subsequently confirmed by repeating reverse transcription and PCR.
  • Unique bands reproducibly displayed in two independent DD-PCR reactions were excised from the dried gel and the cDNA was eluted from the gel by soaking the gel slice in 100 ⁇ l of H 2 O for 10 minutes and subsequent boiling for 15 minutes.
  • the cDNA was recovered by ethanol precipitation in the presence of 3 M NaOAc and 50 ⁇ g glycogen as a carrier and redissolved in 10 ⁇ l of H 2 O.
  • the amplified PCR fragments obtained were analyzed on a 1.5 % agarose gel, then purified using the QUIAquick Gel Extraction kit (Qiagen, Hilden, DE) and used as probes for Northern analysis.
  • Poly A + RNA was isolated from total RNA using the PolyATtract III mRNA Isolation System (Promega Corp., Madison, US). Parallel lanes of poly A + RNA from 4A4 and 2CF cells (1 ⁇ g of each cell line) were size-separated on a denaturing
  • Pre-hybridization (5 hours) and hybridization with radioactive probes overnight were performed in 50 % formamide, 5 x SSC, 5 x Denhardt solution, 1 % SDS and 100 ⁇ g/ml denatured salmon sperm DNA at 42°C.
  • Membranes were washed with 1 x SSC, 0.1 % SDS at room temperature for 15 minutes twice followed by washing with 0.25 x SSC, 0.1 % SDS at 55 to 60°C for 15 to 30 minutes and exposed for autoradiography at -80°C for 48 to 72 hours. Equal loading and transfer of mRNA to the membrane was assessed by hybridizing the blots with P-labeled ⁇ -actin cDNA.
  • Northern analysis was first performed using hybridization probes generated directly from PCR reamplification. Those amplified PCR fragments detecting differentially expressed mRNAs on a Northern blot were subcloned into the PCRII vector by the TA Cloning System (Invitrogen, San Diego, US).
  • Subcloned fragments were isolated using the Qiagen plasmid kit (Qiagen, Hilden, DE) and again used as probes for Northern analysis to verify differential expression.
  • a 450 bp subcloned DD-PCR fragment which detected a differentially expressed mRNA was used as a probe to screen a HeLa cDNA library (Clontech, Palo Alto, US) that had been constructed in a lambda gtlO vector.
  • the isolated cDNA clones were sequenced in a lambda gtlO and compared to the subcloned DD-PCR-fragment.
  • For isolation of full-length cDNA a 5'probe of the cDNA was prepared and used for rescreening of the library. This procedure was repeated for five times until the 5'end of the cDNA was isolated.
  • a 5'RACE (Rapid Amplification of cDNA Ends) PCR was performed following the method as described by Frohmann, M., in PCR Meth. Appl. 4 (1994) 40-48, with some modifications (Schaefer, B., Anal. Biochem. 227 (1995) 255-273).
  • an anti-sense gene-specific 24-mer primer was used for amplification of the 5'-cDNA end.
  • the obtained 5'RACE PCR products were sequenced on both strands and compared to the cDNA clone isolated from cDNA library screening.
  • DRIM tissue-specific expression of DRIM
  • the distribution of DRIM mRNA in different human tissues was analyzed by Northern blot analysis using Multiple Tissue Northern blots (Clontech, Palo Alto, US).
  • the MTN blots containing size-fractionated mRNA extracted from various human tissues were probed with ⁇ P-labeled cDNA probe derived from the 3'-untranslated region of DRIM.
  • Equal loading of mRNA was verified by rehybridizing the MTN blots with
  • both clones are derived from the mammary carcinoma cell line MDA-MB-435 resulting in a metastatic (4A4) and a non-metastatic (2C5) variant.
  • the in vitro growth characteristics of clones 4A4 and 2C5 are almost identical, generally retaining the characteristics of the parent cell line which consists of mononucleated cells with a spindle-shaped appearance, a low cytoplasm to nuclear ratio and visible nucleoli. At confluence around 5 - 10 % of the population consists of multinucleated "giant cells". It is noticed that cell line 2C5 is exhibiting a slightly higher cytoplasm to nucleus ratio. In vivo their behavior is totally different.
  • DRIM mRNA is encoding a new protein.
  • DRIM mRNA Steady state levels of DRIM mRNA in different organs revealed by Northern blotting are displayed in Fig. 3. Strong expression was noted in tissues such as heart, skeletal muscle, pancreas, testis and ovary (Fig. la, f, h. 1 and m). Moderate levels of DRIM mRNA were found in placenta, spleen, thymus " , prostate, small intestine, appendix and fetal liver (Fig. lc, i, j, k, n, p and s). Very low levels of transcripts were identified in brain, colon and bone marrow (Fig. lb, o and r). No DRIM transcripts were found in lung, liver and peripheral blood lymphocytes (Fig. 3d, e and g).
  • DRIM mRNA a broad pattern of expression of DRIM mRNA (Fig. 2) such as in HL60 cells, a promyelocytic leukemia cell line (lane a), Hela cells (lane b), K562 cells derived from chronic myelogenous leukemia (lane c); Raji cells, derived from Burkitt's lymphoma (lane c); SW-480 cells, representing colorectal adenocarcinoma (lane f) and G361 melanoma cells (lane h). Only very weak expression of DRIM mRNA was noted in MOLT4 cells representing lymphoblastic leukemia (lane d) and lung carcinoma A549 cells (lane g)-
  • DRIM transcript levels were scored in several mammary carcinoma cell lines (Fig. 3).
  • DRIM cDNA was cloned as a lO kb Notl fragment into the Notl site of the mammalian cell expression vector pcDNA3.1 (Invitrogen, Order No. V790-20) and the resulting plasmid isolated according to standard methods.
  • the resulting plasmid is referred to as pcDNA3.1 DRIM.
  • 10 ⁇ g of pcDNA3.1 DRIM and, separately, 10 ⁇ g of insert-free vector pcDNA3.1 were transfected into the metastatic subclone 4A4 by the calcium-phosphate method (Graham and van der Eb, Virology 52 (1973) 456-463) and stable transfectants isolated at 800 ⁇ g/ml of G418.
  • Stable clones were isolated and grown up to mass culture. Metastatic capacity of 5 clones transfected with pcDNA3.1 DRIM, 4 clones transfected with the insert-free vector pcDNA3.1 as well as the metastatic cell line 4A4 were assessed by intravenous injection of 1x10 cells in 0.1 ml serum-free DMEM into the lateral tail vein of nude mice. Recipient animals were killed and autopsied 2 months after injections.

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Abstract

L'invention concerne un nouveau gène, DRIM, ainsi que son produit génique. L'invention concerne également une méthode d'isolement de ce produit génique, et l'utilisation de ce produit génique pour obtenir un agent thérapeutique. Les séquences d'ADN et les séquences protéiques sont illustrées dans SEQ ID NOS:1 et 2. Le gène DRIM de cette invention présente une activité antimétastatique.
PCT/EP1999/003396 1998-05-18 1999-05-17 Acide nucleique a regulation negative de la metastase (drim) codant pour une proteine, et utilisation diagnostique et therapeutique de cet acide nucleique Ceased WO1999060116A1 (fr)

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AU42635/99A AU4263599A (en) 1998-05-18 1999-05-17 Nucleic acid encoding protein, down-regulated in metastasis (drim), and its diagnostic and therapeutic use

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EP98303895 1998-05-18
EP98303895.1 1998-05-18

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WO1999060116A1 true WO1999060116A1 (fr) 1999-11-25

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PCT/EP1999/003396 Ceased WO1999060116A1 (fr) 1998-05-18 1999-05-17 Acide nucleique a regulation negative de la metastase (drim) codant pour une proteine, et utilisation diagnostique et therapeutique de cet acide nucleique

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996039504A2 (fr) * 1995-06-06 1996-12-12 Novartis Ag Procede permettant la distinction entre cancers metastatiques et cancers non metastatiques, et polynucleotides et polypeptides utilises dans ce procede

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996039504A2 (fr) * 1995-06-06 1996-12-12 Novartis Ag Procede permettant la distinction entre cancers metastatiques et cancers non metastatiques, et polynucleotides et polypeptides utilises dans ce procede

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HILLIER L ET AL: "Homo sapiens cDNA clone 550225 (accession number AA085514)", EMBL DATABASE, 24 October 1996 (1996-10-24), Heidelberg, Germany, XP002081969 *
SCHWIRZKE M ET AL: "Differential gene expression in mammary carcinoma cell lines: identification of DRIM, a new gene down-regulated in metastasis", ANTICANCER RESEARCH, vol. 18, May 1998 (1998-05-01), pages 1409 - 1422, XP002081970 *

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