WO1986003226A1 - Genes transmissibles de mammiferes associes a des metastases de tumeurs - Google Patents

Genes transmissibles de mammiferes associes a des metastases de tumeurs Download PDF

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WO1986003226A1
WO1986003226A1 PCT/US1985/002323 US8502323W WO8603226A1 WO 1986003226 A1 WO1986003226 A1 WO 1986003226A1 US 8502323 W US8502323 W US 8502323W WO 8603226 A1 WO8603226 A1 WO 8603226A1
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cells
metastasis
dna
tumor
marker
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PCT/US1985/002323
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English (en)
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Shelly C. Bernstein
Robert A. Weinberg
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Whitehead Institute For Biomedical Research
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Priority to HU8667A priority Critical patent/HUT41837A/hu
Publication of WO1986003226A1 publication Critical patent/WO1986003226A1/fr
Priority to DK359486A priority patent/DK359486A/da
Priority to FI863095A priority patent/FI863095A/fi
Priority to NO863066A priority patent/NO863066L/no

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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
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • G01N33/5017—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity for testing neoplastic activity
    • 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
    • C12Q2600/118—Prognosis of disease development

Definitions

  • This invention is in the field of molecular biology and more specifically relates to the detec ⁇ tion and isolation of discrete, transmissible, ma- alian genes associated with tumor metastasis.
  • Oncogenes such as those of the ras group, are able to induce full tumorigenic conversion of immortalized cells, such as NIH 3T3 mouse fibro- blasts.
  • immortalized cells such as NIH 3T3 mouse fibro- blasts.
  • the effects of these ras oncogenes on primary embryo fibroblasts are more circumscribed; in these cells, the ras oncogene requires the collaboration of a second oncogene, such as myc to induce tumorigenicity.
  • This invention relates to the discovery of a discrete, transmissible, mammalian gene associated with tumor metastasis. '
  • One embodiment of the invention comprises a method for isolating such a discrete, transmissible gene associated with tumor metastasis from the DNA of a mammalian species.
  • donor DNA from metastatic mammalian tumor cells
  • the multiplicity of fragments and a selectable marker are then trans ⁇ mitted into recipient cells capable of phenotypi- cally expressing the presence of the selectable marker as well as the discrete, transmissible gene of interest.
  • the recipient cells are then cultured under conditions which allow phenotypic expression of the selectable marker.
  • recipient cells can be selected which have acquired the gene of interest, in addi ⁇ tion to the marker; they may also contain-additional DNA sequences from the donor fragments transmitted into the recipient cells, as well as their own endogenous DNA.
  • Recipient cells selected because of phenotypic expression of the selectable marker are introduced into a mammalian host under conditions such that the introduced cells would not be meta ⁇ static in the absence of the acquired gene of interest. These cells are allowed to form a primary tumor at the site of introduction in the mammalian host, and the host is then examined for metastases. DNA is then recovered from cells of any metastases formed in the host or from cultures of cells removed from the metastases.
  • DNA recovered from such metastasis is then broken into fragments, at least one of which is a fragment containing essentially only the gene of interest and donor marker, after which the gene of interest is re ⁇ covered.
  • Such recovery may be, for example, by procedures of gene cloning.
  • a gene associated with metastasis of the human cell line ME-180 a human cervical carcinoma metastatic to omenturn
  • the original DNA from this cell line was serially cotransfected into NIH 3T3 mouse fibroblast cells with pSV2neo DNA, a selectable marker. Subcutaneous injection of such cells into immunocompetent mice resulted in an initial metastasis to the lung of one mouse and a subsequent metastasis to the abdomen of a second mouse.
  • the ME180 gene does not appear to be closely related to the royc oncogene or to the ras oncogenes in that it did not exhibit reactivity in nucleic acid hybridization tests using DNA probes that are specific for the yc or ras oncogenes.
  • a gene associated with metastasis of the human cell line SK-N-MC a human neuroblastoma metastatic to the supraorbital area
  • SK-N-MC a human neuroblastoma metastatic to the supraorbital area
  • protein coded for by the gene can be produced in significant quantity so that it can be studied to understand the metabolic alterations that occur in the cell during the expression of the metastatic trait. This may also lead to insights into methods by which such a gene, gene products or cellular products affected by the gene product could be antagonized or inhibited. It is, of course, expected to lead to sensitive tests for the presence of this gene or products of this gene involving probes for the gene, RNA transcribed from the gene, or antibodies reactive with products of the gene.
  • The" figure is a photograph of a blot illus ⁇ trating the results of tests for the presence of human ras and myc oncogene sequences in DNA from metastases derived from secondary transfectants of EJ-6-2-Bam-6a cells.
  • any discrete, transmissible mammalian gene associated with meta ⁇ stasis could be detected using the techniques of this invention.
  • a "discrete” gene is one having a contiguous sequence of base pairs located in one bloc__'of sequences of definable length.' This block may contain regions coding for protein as well as intervening sequence regions which do not encode protein.
  • a "transmissible” gene is one which can be transmitted from cell to cell using gene transfer (transfection) techniques.
  • Donor DNA containing such a discrete, trans ⁇ missible gene can be isolated from other host cell constituents by art-recognized techniques. For example, cells grown in culture can be lysed and the viscous lysate can then be extracted with phenol and with chloroform-isoamyl alcohol. DNA can then be precipitated by ethanol precipitation.
  • the initial donor DNA containing the gene of interest can be fragmented by mechanical or enzy ⁇ maticmethods. For example, it can be passed through a narrow gauged needle so that it will be fragmented due to shear. On the other hand, DNA molecules might also be fragmented using restriction endonucleases. The important limitation on frag- mentation is that at least one fragment results each time the fragmentation is done which contains in intact form all of the gene of interest.
  • a donor cell may be tagged with copies of cloned DNA sequence, such as a 0X174 bacteriophage DNA fragment. Upon co-transfection, the donor cell DNA fragments and the cloned marker or "tag" DNA fragments become linked in the recipient cell in a randomly alternating co-polymer. See Lowy, I., Pellicer, A., Jackson, J.F., Simas, I.M. G. K., Silverstein, S. and Axel, R. (1980) Cell 22; 817-823.
  • the fragment containing the gene of interest will contain a naturally associated donor-specific marker.
  • human DNA contains over 300,000 copies of the Alu sequence interspersed throughout the entire genome which can serve as a specific marker indicating the presence of human DNA segments.
  • almost every gene is linked closely, i.e., less than 10 kilobases, to a copy of this repeated sequence which can serve as marker. See Houck, CM., Rinehart, F.D., and Sch id, C.W. (1979) J ⁇ Mol. Biol. 132; 289-306. Since this Alu sequence is not present in mouse DNA, it is species-specific to human DNA.
  • fragmented donor DNA can be transmitted into cells with a selectable marker which can be phenotypically expressed by the cells.
  • the selectable marker employed was the plasmid pSV2neo. Subjecting cultures to G418 selection kills all cells except those carrying this plasmid because of cytotoxic effects of the G418 drug, which drug is inactivated in cells that have acquired the pSV2neo gene. See Southern, E.M. and Berg, P. (1982) J. Mol. Appl. Genet. 1; 327-341.
  • selectable markers could be employed such as the Ecogpt marker which confers resistance to the cytostatic effects of the drug mycophenolic acid.
  • the preferred route for transmitting donor DNA fragments and the selectable marker into cells is transfection, such as calcium phosphate facilitated transfection.
  • DNAs of any sequence or biological origin can be introduced into mammalian cells by transfection.
  • the transfected DNA need not have any sequence homology with the genome of the recipient cell.
  • Co-transfection is the co-mingling of two or more DNAs prior to their being introduced into mammalian cells, such as by the calcium phosphate facilitated transfection procedure.
  • EJ-6-2-Bam-6a cells were employed as recipients for the genes associated with metastasis in the experiments described herein.
  • Such cells are NIH 3T3 mouse fibroblast cells transformed with the EJ-Ha-ras oncogene, whose presence allows these cells to fpr tumors which are localized and non- metastatic. Furthermore, these cells contain only the 6.6 kb fragment of the EJ-Ha-ras oncogene, devoid of any Alu sequences.
  • NIH 3T3 cells, and their derivatives, are unusual because they take up and allow efficient stable expression of transfected DNAs.
  • Rat-1 cell line Another suitable cell line for transfection is known as Rat-1 cell line. Although most cell lines are relatively refractory to transfected DNAs, it is believed that other cell lines will be found which allow expression of transfected genes.
  • co-transfected cells After co-transfected cells have phenotypically expressed the presence of the selectable marker, cells showing such expression are selected. For example, a colony expressing the phenotype can be physically picked by standard techniques and the cells of the chosen colony can then be used to seed a large scale culture. Those cells which have expressed the selectable marker can be shown to' have concomitantly acquired substantial amounts of the donor cell genomic DNA whose presence may not be manifested in phenotypic alterations of these cells that are apparent in monolayer culture.
  • Such selected recipient cells can then be introduced into a mammalian host under conditions such that these recipient cells would be tumorigenic but not be metastatic unless they have acquired a donor gene associated with tumor metastasis.
  • Subcutaneous injection is one suitable technique, but the experimental work described herein also documents that intramuscular injection, intraperi- toneal inoculation as well as injection into the foot pad of mice were suitable also. Intravenous injection was not suitable since otherwise non- metastasizing tumors created metastases when the tumor cells were intravenously injected.
  • DNA is then recovered from metastatic cells of metastases formed in the host and employed in place of the original mammalian donor DNA for a second round of transfection into the tumorigenic non-meta- static EJ-6-2-Bam-6a cells.
  • the preceding steps are repeated, as required. By repeating all of these steps, serial transmission of the gene of interest, together with any donor marker sequence, are accomplished.
  • a marker gene may be a human repetitive DNA sequence of the Alu type such as that which could be detected by use of the BLUR probe; alternatively, the gene of interest could be experimentally linked to a marker gene prior to the first of the two transfections and this marker gene could then be identified by use of a cloned probe that is reactive with this marker gene. Recovery of the gene can be done by creating- a genomic library.
  • the gene of interest or a portion of it may be contained within a phage also carrying a donor specific marker sequence such as an Alu sequence or an experi ⁇ mentally added marker. This phage may be identified employing standard hybridization procedures and the phage can be amplified, yielding the human DNA segment in cloned form.
  • the mammalian gene associated with the Alu sequence can be further cloned into other suitable recombinant DNA vectors.
  • suitable recombinant DNA vectors include bacterial plasmids, phages, animal viruses and yeast vectors. In such techniques, hosts would be employed which allow the recombinant DNA vector to multiply.
  • Such proteins can be studied and their effects on modifying other cellular constituents and regulating their level and activity can be eluci ⁇ dated. Moreover, such proteins can be used to produce antibodies by standard antibody production techniques. Thus, for producing polyclonal anti ⁇ bodies, such proteins would be employed to immunize a host, such as a rabbit or a rat, and antibodies to the protein would be collected from serum obtained from the host.
  • monoclonal antibodies could be produced employing cells which produce antibodies to the protein produced by the isolated gene in typical fusion techniques for forming hybridoma cells. Basically, these techniques involve the fusing of the antibody producing cell with a cell having immortality, such as a myeloma cell, to provide a fused cell hybrid which has immortality and is capable of producing the desired antibody, in this case an antibody to the protein coded for by the isolated gene. The hybrid cells are then cultured under conditions conducive to the production of antibody which is subsequently collected from the cell culture medium.
  • Such techniques for producing monoclonal antibodies have been well described in the literature. See, for example, U.S. Patent Nos. 4,172,124 and 4,196,265 issued to Hilary Koprowski et al.
  • a significant use for the antibodies produced , to such protein is in assays to detect the presence of protein coded for by the isolated gene associated with metastasis.
  • assays include immunoassays, such as those radioimmunoassays employing labelled antibodies or viral antigens.
  • Probes could also be employed for detecting the gene associated with metastasis or mRNA transcribed by said gene.
  • Such probes might comprise, for example, a labelled polynucleotide complementary to at least a portion of a mammalian gene associated with tumor metastasis.
  • Metastasis may be prevented or dimininshed by employing antibodies capable of neutralizing protein expressed by a mammalian gene associated with metastasis of the tumor.
  • metastasis may be prevented or diminished by antagonizing the gene associated with metastasis, or by antagonizing any cellular product or intermediate affected by protein expressed by said gene.
  • the EJ transfectant termed EJ-6-2-Bam-6-a, was an NIH 3T3-derived tertiary transfectant carrying the EJ-Ha-ras bladder carcinoma oncogene. It was obtained by passing DNA by transfection in two serial cycles through NIH 3T3 cells. DNA of the secondary transfectant was cleaved with the restric ⁇ tion endonuclease Bam HI prior to the third cycle of transfection, freeing the Ha-ras oncogene from linkage to the human Alu repeat sequences.
  • mice were inoculated into either immuno- competent histocompatible mice, which were 6-8 weeks old NFS/NCr mice or immunoincompetent mice, which were NIH nude (nu/nu) mice previously irradiated with 500 rad.
  • Each mouse was inoculated by inject- ing subcutaneously into the flanks of the mice 10 cells in 0.2 ml phosphate buffered saline. The results were as follows:
  • tumorigen ⁇ icity The ability of the inoculated cells to form tumors at the site of inoculation, termed tumorigen ⁇ icity, was determined by making a ratio of the number of mice in which tumors grew over the number of mice inoculated. Metastatic ability of the tumors was rated by making a ratio of the number of mice found to have metastases over the number of mice found to have tumors at the site of inocula- tion. Metastasis was determined by sacrificing moribund mice up to 6 weeks after inoculation and subjecting them to necropsy.
  • NIH 3T3 cells were cotransfected with normal cellular DNA from NIH 3T3 cells and pSV2neo plasmid DNA following the procedures of Andersson e_t al. See Andersson, P., Goldfarb, M.P., and Weinberg, R.A. (1979) Cell 16; 63-75.
  • NIH 3T3 DNA and 1 ug of pSV2neo plasmid DNA were applied to 7.5 x 10 5 EJ-transformed NIH 3T3 cells, EJ-6-2- Bam-6-a (2 x 10 cm dishes) . Cells were split in a ratio of 1:6. The following day, the cultures were subjected to G418 selection, which killed all cells except those carrying the pSV2neo DNA. See South- ern, E.M. and Berg, P. (1982) J. Mol. Appl. Genet. 1_: 327-341. Colonies resistant to G418 were counted 14 days after transfection.
  • the G418-resistant cells were collected after they grew to form a dense confluent monolayer
  • mice inoculated intramuscularly in the foot pad or intraperitoneally did not form metastases.
  • mice intravenously injected in the lateral tail vein formed lung metastases without the presence of a primary tfcmor, this did not bear on subsequent experiments, all of which employed injection by a subcutaneous route.
  • Examples 1 and 2 when subcutaneously injected into mice and, being of NIH 3T3 origin, were able to take up efficiently and express exogenous DNA. See Smotkin, D. , Gianni, A.M., Rozenblatt, S. and Weinberg, R.A. (1975) Proc. Natl. Acad. Sci. USA 72: 4910-4913. DNAs from a variety of human metastatic tumor cell lines were applied to cultures of the Ha-ras transformant (EJ-6-2-Bam-6a) , using the cotransfec- tion procedure of Example 2 except substituting- a variety of human metastatic tumor cell line DNAs for the normal NIH 3T3 cell line DNA.
  • EJ-6-2-Bam-6a Ha-ras transformant
  • Human metastatic cell lines whose DNAs were employed included: ME-180, human cervical carcinoma, metastatic to omentum; SK-N-MC, human neuroblastoma, metastatic to supraorbital soft tissue; HuTu 80, human metastatic duodenal adenocarcinoma; Capan-1, human pancreatic adenocarcinoma, metastatic to liver; Calu-1, human lung carcinoma, metastatic to pleura; SK-N-SH, human neuroblastoma, metastatic to bone marrow; Hs0895, human melanoma, metastatic to lung; Hs0891, human renal cell carcinoma, metastatic to lymph node; and
  • SK-MEL-5 human melanoma, metastatic to lymph node.
  • the metastatic human tumor DNA was cotransfected with pSV2neo plasmid and G418 selec ⁇ tion was applied to the transfected cultures. All untransfected cells were thus killed which allowed the outgrowth of large numbers of colonies carrying the pSV2neo marker and a substantial amount of concomitantly acquired human tumor DNA.
  • mice Nine of the resulting cultures were inoculated into NFS/NCr mice employing the inoculation procedures of Example 3; six mice has large tumors. Upon autopsy, 4 of the 6 mice carried extensive metastases that were detect- able 14-21 days after inoculation.
  • DNAs from 2 of the 4 metastases arising from secondary transfectants were cotransfected once again with pSV2neo DNA and a third cycle of transfection and the resulting cultures tested for metastatic ability in NFS/NCr mice.
  • One secondary transfectant yielded DNA which was used to induce a group of tertiary transfectants.
  • These tertiary transfectants induced primary tumors in 18 of 22 mice inoculated and metastasis was observed in 8 of the 18 mice having primary tumors.
  • the derived tertiary transfectants resulted in primary tumors in 11 of 12 mice inoculated and metastasis was observed in 3 of the 11 mice having these primary tumors. Histological examination of the pulmonary and intraperitoneal metastases revealed that these metastatic tumors were fibrosarcomas, as were the primary non-metasizing tumors.
  • DNA was also prepared from the second metastasis derived from primary transfectants of human ME180 DNA and cotransfected into Ha-ras transformed NIH 3T3 cells (EJ-6-2-Bam-6a) with pSV2neo employing the procedures of Example 3.
  • the culture of these cells was divided into 12 sub ⁇ cultures after one day and then placed under G148 selection. The cultures were inoculated into
  • NFS/NCr mice employing the inoculation procedures of Example 3. All twelve mice had evidence of tumor formation; six of the twelve mice carried extensive metasases detectable upon autopsy 14-21 days after inoculation.
  • DNA was prepared from the single metasasis derived from primary transfectants of human SK-N-MC DNA and co-transfected • into Ha-ras transformed NIH 3T3 cells (EJ-6-2-Bam-6a) with pSV2neo employing the procedures of Example 3.
  • the culture of these cells was divided into 12 sub- cultures after one day and then placted under G148 selection.
  • the cultures were inoculated into NFS/NCr mice employing the inoculation procedures of Example 3. All twelve mice had evidence of tumor formation; seven of the twelve mice carried exten ⁇ sive metastases detectable upon autopsy 14-21 days after inoculation.
  • the DNAs analyzed here are as follows: a_, ⁇ , £, j_, EJ-6-2-Bam-6a tertiary trans ⁇ fectant derived from EJ bladder carcinoma DNA; lanes __.' ' ii' £' ME180-2°Metl2L, metastasis derived from secondary transfectants of EJ-6-2-Bam-6a cells transfected with ME-180 metastatic carcinoma DNA; lanes £, _f, i_, _1, ME-180 human metastatic cervical carcinoma. Size markers (in kilobases) are indi ⁇ cated on the ordinate.
  • DNAs from two of the four metastases derived from the secondary transfectants were digested with the restriction endonucleases EcoRI, BamHI, and Hindlll. Digested DNAs were religated with genomic NIH 3T3 DNA. The resulting DNAs were then co ⁇ transfected with pSV2neo DNA into EJ-6-2-Bam-6 ' a cells and placed under G418 selection. Cells selected were inoculated subcutaneously into NFS/NCr mice (10 cells/mouse) . Two to six weeks later, mice were examined for metastasis.
  • the invention described herein is useful in detecting and isolating discrete, transmissible mammalian genes associated with tumor metastasis. Detection and isolation of such genes makes it possible to develop probes for such genes, or mRNA transcribed from such genes, as well as the produc ⁇ tion of antibodies against proteins encoded by such genes. This allows sensitive diagnostic techniques for metastasis and potential antagonism of such genes, gene products, or cellular products or intermediates affected by such gene products.

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Abstract

Un procédé permet de détecter un gène isolé et transmissible d'un mammifère, associé à la métastase de tumeurs. On a détecté et identifié par ce procédé un gène associé à la métastase et originaire de la lignée ME-180 du cancer cervical humain, provoquant des métastases de l'épiploon de même qu'un gène associé à la métastase et originaire de la lignée SK-N-MC des cellules humaines de neuroblastomes, provoquant des métastases de la zone sus-orbitaire.
PCT/US1985/002323 1984-11-30 1985-11-26 Genes transmissibles de mammiferes associes a des metastases de tumeurs WO1986003226A1 (fr)

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Application Number Priority Date Filing Date Title
HU8667A HUT41837A (en) 1984-11-30 1985-11-26 Transformable genes of mammals causing tumoral metastasis
DK359486A DK359486A (da) 1984-11-30 1986-07-29 Overfoerbare pattedyrgener forbundet med tumormetastase, fremg. til isolering deraf, proteiner som generne koder for, antistoffer over for disse, sonder for generne og for mrna transkriberet derfra og fremg. til bedoemmelse af tumorers metastatiske tilboejelighed
FI863095A FI863095A (fi) 1984-11-30 1986-07-29 Transmissibla daeggdjursgener associerade med tumoermetastaser.
NO863066A NO863066L (no) 1984-11-30 1986-07-29 Overfoerbare pattedyrgener som er forbundet med tumormetastase.

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US67662584A 1984-11-30 1984-11-30
US676,625 1991-03-28

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JP (1) JPS62501399A (fr)
AU (1) AU5197986A (fr)
DK (1) DK359486A (fr)
ES (1) ES8705043A1 (fr)
FI (1) FI863095A (fr)
GR (1) GR852864B (fr)
HU (1) HUT41837A (fr)
IL (1) IL77157A0 (fr)
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US7462491B2 (en) 2002-01-31 2008-12-09 Baylor College Of Medicine Methods and compositions for diagnosis and monitoring of prostate cancer progression by detection of serum caveolin

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FI863095A0 (fi) 1986-07-29
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NO863066D0 (no) 1986-07-29
PT81584B (en) 1987-04-07
DK359486D0 (da) 1986-07-29
FI863095A (fi) 1986-07-29
ES549438A0 (es) 1987-04-16
GR852864B (fr) 1986-03-28
EP0203970A1 (fr) 1986-12-10
HUT41837A (en) 1987-05-28
NO863066L (no) 1986-09-29
AU5197986A (en) 1986-06-18
PT81584A (en) 1985-12-01
ES8705043A1 (es) 1987-04-16
DK359486A (da) 1986-07-29
IL77157A0 (en) 1986-04-29

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