Classifications

• • 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

Definitions

• the present invention relates to gene expression in normal cells and cells of metastasizing tumors and particularly to a novel metastasis-associated antigen, C4.4A, and the gene encoding C4.4A.
• Tumor progression is a complex process, which involves detachment from the primary tumor, migration through the extracellular matrix, penetration through the basal membrane, adaptation to the circulation pressure, attachment to the endothelia of the vessel wall and settlement and growth in distant organs.
• metastatic cells frequently display a whole array of qualitatively or quantitatively altered gene products. These include, as the most frequent ones, cell-cell and cell-matrix adhesion molecules as well as matrix-degrading enzymes, their activators, inhibitors and receptors.
• pancreatic adenocarcinoma of the rat which metastasizes via the lymphatic system and expresses a variety of surface molecules, which are also expressed on non-related, metastasizing tumor cells, but never an non-metastasizing tumors.
• all of these molecules were detected under physiological conditions, whereby the expression pattern varied between widespread distribution and restriction to few selected tissue layers. All of these molecules were expressed or upregulated during implantation and placentation. Some, though not all were differentially expressed during organogenesis.
• One of these molecules has been identified as a variant isoform of the adhesion molecule CD44.
• D6.1A is a tetraspanin molecule and the homologue of the human tumor antigen CO-029.
• the molecule is widely expressed and there is evidence that its metastasis-associated function is brought about by its linkage to additional surface structures, particularly integrins .
• a molecule, D5.7A, is the homologue to the human panepithelial antigen EGP314, whose function in tumor progression is currently under investigation.
• Matrix degrading enzymes like matrix metalloproteinases (MMP) and urokinase (uPA) are known to play an important role in metastasis formation and much effort has been put into trials to prevent activation of such enzymes as a therapeutic regimen.
• MMP matrix metalloproteinases
• uPA urokinase
• these enzymes can also be found in normal tissue. Thus, they are not useful as markers specific for metastasizing tumors and, even more importantly, the inhibition of the activity or expression of these enzymes for tumor therapy might be accompanied by severe side effects.
• the present invention is based on the isolation of a gene encoding a novel metastasis-associated antigen, C4.4A, particularly the rat and human C4.4A. Both antigens are expressed under physiological conditions only in the gravid uterus and on epithelial cells of the upper gastrointestinal tract.
• the rat cDNA of the antigen (1.637 bp) codes for a 352 amino acid long glycosylphosphatidyl-inositol (GPI) anchored molecule, whose molecular weight varies in different cells between 94 - 98 kDa according to the degree of N- and 0- glycosylation.
• the length of the human C4.4A is 347 amino acids.
• C4.4A exerts functional activity similar to uPAR, i.e. via activation of matrix degrading enzymes.
• uPAR matrix degrading enzymes
• C4.4A exerts functional activity similar to uPAR, i.e. via activation of matrix degrading enzymes.
• C4.4A exerts functional activity similar to uPAR, i.e. via activation of matrix degrading enzymes.
• C4.4A exerts functional activity similar to uPAR, i.e. via activation of matrix degrading enzymes.
• C4.4A e.g. the inhibition of its expression or activity
• the C4.4A molecule is an important target for screening, e.g. for diagnostic purposes.
• the present invention thus, provides a C4.4A protein as well as a nucleic acid molecule encoding the protein and, moreover, an antisense RNA, a ribozyme and an inhibitor, which allow to inhibit the expression or the acitivity of C4.4A.
• the present invention provides a diagnostic method for detecting a metastasizing tumor associated with C4.4A in a tissue of a subject, comprising contacting a sample containing C4.4A or C4.4A encoding mRNA with a reagent which detects C4.4A or the corresponding mRNA.
• the present invention provides a method of treating a metastasizing tumor associated with C4.4A, comprising administering to a subject with such an disorder a therapeutically effect amount of a reagent which modulates, e.g. inhibits, C4.4A expression or the activity of the protein, e.g. the above described compounds.
• the present invention provides a method of gene therapy comprising introducing into cells of a subject an expression vector comprising a nucleoitde sequence encoding the above mentioned antisense RNA or ribozyme, in operable linkage with a promotor.
• Figure 1 Nucleic acid sequence and deduced amino acid sequence of the rat C4.4A
• Figure 2 Nucleic acid sequence and deduced amino acid sequence of the human C4.4A
• Figure 3 Molecular weight of the rat C4.4A protein and mRNA
• C4.4A cDNA COS-7 C4.4
• COS-7 D6.1 COS-7 D6.1
• C4.4A molecule is expressed in BSp73ASML, Regressor and C4.4A cDNA transfected COS-7 cells.
• the molecular weight varies between BSp73ASML (98 kDa) and RG as well as COS-7 C4.4
• the molecular weight of C4.4A cDNA transfected AS cells was not changed by inhibiting O-glycosylation, the molecular weight of C4.4A on BSp73ASML was reduced to 94 kDa corresponding to the molecular weight of C4.4A on PROG.
• Paraffin sections through lung metastases of BSp73AS-mock (a) , BSp73AS-lBl (b) , BSp73AS-2A2 (c) , BSp73AS-lBl (higher magnification) (d) and BSp6S-C4.4A (e) are shown.
• lung metastases of rat C4.4A transfected tumors are not encapsulated (closed arrow head) (b, c and e) and degrade the bronchus epithelium (closed arrow) (e) and the vessel endothelium (open arrow) (d) .
• the tumor lines PROG, 73ASML, 73AS-mock and 73AS-1B1 were labeled with 51 Cr and were seeded (5 x 10 4 cells/well) on BSA or laminin coated plates. Where indicated, the medium contained 10 ⁇ g/ml control IgGl and C4.4 , respectively. Cells were incubated for 30 - 120 min. Non-adherent cells were washed off and adherent cells were detached by trypsin treatment. Radioactivity was determined in a ⁇ -counter. Mean c.p.m. +/- s.d. of triplicate cultures are shown, (a) Cells were incubated in RPMI/5% FCS for 30 min. and 120 min. at 37°C on BSA or laminin coated plates, the medium containing either
• FISH fluorescence in situ hybridization
• a Northern blot was probed with C4.4A as described in Example 1.
• Each lane represents 2 ⁇ g of poly (A) -RNA of brain, placenta, lung, liver, skeletal muscle, kidney and pancreas.
• the lower panel shows the GAPDH control
• b Expression of C4.4A on non-transformed tissues analyzed by RT-PCR. RT-PCR probes derived from poly A RNA were separated on a 1% agarose gel. First and last lane: 100 bp ladder, lane 2: negative control (H 2 0) , lane 3-14: tissue samples, lane 15-17: three positive controls with the depicted copy numbers of hC4.4A derived from a plasmid.
• the lower panel shows the GAPDH control .
• the present invention relates to isolated nucleic acid molecule encoding the metastasis-associated antigen C4.4A, particularly the rat and human C4.4A, or a protein exhibiting biological properties of the metastasis-associated antigen
• nucleic acid molecule which represents a fragment, derivative or allelic variant of a nucleic acid sequence specified in (a) to (e) .
• a protein exhibiting biological properties of metastasis-associated antigen C4.4A is understood to be a protein having at least one of the acitivities of C4.4A as illustrated in the Examples, below.
• isolated nucleic acid molecule includes nucleic acid molecules substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which it is naturally associated.
• the invention provides an isolated nucleic acid molecule encoding the metastasis-associated antigen C4.4A comprising the amino acid sequence depicted in Figure 1 and 2, respectively.
• the present innvention also provides a nucleic acid molecule comprising the nucleotide sequence depicted in Figure 1 and 2, respectively.
• the present invention provides not only the generated nucleotide sequence identified in Figure 1 and 2, respectively and the predicted translated amino acid sequence shown in Figure 1 and 2, respectively, but also plasmid DNA containing a DNA C4.4A (rat) deposited with the DSMZ on 18.08.1999 under DSM 13013 and a DNA C4.4A (human) deposited with the DSMZ on 18.08.1999 under DSM 13014, respectively.
• the nucleotide sequence of each deposited C4.4A clone can readily be determined by sequencing the deposited clone in accordance with known methods. The predicted C4.4A amino acid sequence can then be verified from such deposits.
• amino acid sequence of the protein encoded by each deposited clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited C4. A encoding DNA; collecting the protein, and determining its sequence.
• the nucleic acid molecules of the invention can be both DNA and RNA molecules. Suitable DNA molecules are, for example, genomic or cDNA molecules. It is understood that all nucleic acid molecules encoding all or a portion of C4.4A are also included, as long as they encode a polypeptide with C4.4A activity.
• the nucleic acid molecules of the invention can be isolated from natural sources or can be synthesized according to known methods .
• the present invention also provides nucleic acid molecules which hybridize to the above nucleic acid molecules.
• hybridize has the meaning of hybridization under conventional hybridization conditions, preferably under stringent conditions as described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2 nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
• nucleic acid molecules that hybridize to the C4.4A nucleic acid molecules at lower stringency hybridization conditions Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency) ; salt conditions, or temperature.
• washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC) .
• Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
• Nucleic acid molecules that hybridize to the molecules of the invention can be isolated, e.g., from genomic or cDNA libraries that were produced from rat or human cell lines or tissues. In order to identify and isolate such nucleic acid molecules the molecules of the invention or parts of these molecules or the reverse complements of these molecules can be used, for example by means of hybridization according to conventional methods (see, e.g., Sambrook et al . , 1989, Molecular Cloning, A Laboratory Manual, 2 nd edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) . As a hybridization probe nucleic acid molecules can be used, for example, that have exactly or basically the nucleotide sequence depicted in Figure 1 and 2, respectively, or parts of these sequences. The fragments used as hybridization probe can be synthetic fragments that were produced by means of conventional synthesis methods and the sequence of which basically corresponds to the sequence of a nucleic acid molecule of the invention.
• the nucleic acid molecules of the present invention also include molecules with sequences that are degenerate as a result of the genetic code.
• the present invention provides nucleic acid molecules which comprise fragments, derivatives and allelic variants of the nucleic acid molecules described above encoding a protein of the invention.
• “Fragments” are understood to be parts of the nucleic acid molecules that are long enough to encode one of the described proteins.
• the term “derivative” in this context means that the sequences of these molecules differ from the sequences of the nucleic acid molecules described above at one or several positions but have a high level of homology to these sequences .
• Homology hereby means a sequence identity of at least 40 %, in particular an identity of at least 60 %, preferably of more than 80 % and particularly preferred of more than 90 %.
• nucleic acid molecules have a sequence identity to the amino acid sequence depicted in Figure 1 and 2, respectively, of at least 80 %, preferably of 85 % and particularly preferred of more than 90 %, 95 %, 97 % and 99 %.
• the deviations to the above-described nucleic acid molecules may have been produced by deletion, substitution, insertion or recombination .
• nucleic acid molecules that are homologous to the above- described molecules and that represent derivatives of these molecules usually are variations of these molecules that represent modifications having the same biological function. They can be naturally occurring variations, for example sequences from other organisms, or mutations that can either occur naturally or that have been introduced by specific mutagenesis. Furthermore, the variations can be synthetically produced sequences.
• allelic variants can be either naturally occurring variants or synthetically produced variants or variants produced by recombinant DNA processes.
• muteins can be produced, for example, that possess a modified K,.-value or that are no longer subject to the regulation mechanisms that normally exist in the cell, e.g. with regard to allosteric regulation or covalent modification. Such muteins might also be valuable as therapeutically useful antagonists of C4.4A.
• nucleic acid molecules of the invention or parts of these molecules can be introduced into plasmids allowing a mutagenesis or a modification of a sequence by recombination of DNA sequences .
• bases can be exchanged and natural or synthetic sequences can be added.
• natural or synthetic sequences can be added.
• manipulations can be performed that provide suitable cleavage sites or that remove superfluous DNA or cleavage sites. If insertions, deletions or substitutions are possible, in vitro mutagenesis, primer repair, restriction or ligation can be performed.
• analysis method usually sequence analysis, restriction analysis and other biochemical or molecular biological methods are used.
• the proteins encoded by the various variants of the nucleic acid molecules of the invention show certain common characteristics, such as enzyme activity, molecular weight, immunological reactivity or conformation or physical properties like the e 1 e c t o rphor e t i c a 1 mobility, chromatographic behavior, sedimenta ion coefficients, solubility, spectroscopic properties, stability; pH optimum, temperature optimum.
• the invention furthermore relates to vectors containing the nucleic acid molecules of the invention.
• they are plasmids, cosmids, viruses, bacteriophages and other vectors usually used in the field of genetic engineering.
• Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria, the pMSXND expression vector for expression in mammalian cells and baculovirus-derived vectors for expression in insect cells.
• the nucleic acid molecule of the invention is operatively linked to the regulatory elements in the recombinant vector of the invention that guarantee the transcription and synthesis of an RNA in prokaryotic and/or eukaryotic cells that can be translated.
• the nucleotide sequence to be transcribed can be operably linked to a promotor like a T7 , metallothionein I or polyhedrin promotor.
• the present invention relates to recombinant host cells transiently or stably containing the nucleic acid molecules or vectors of the invention.
• a host cell is understood to be an organism that is capable to take up in vi tro recombinant DNA and, if the case may be, to synthesize the proteins encoded by the nucleic acid molecules of the invention.
• these cells are prokaryotic or eukaryotic cells, for example mammalian cells, bacterial cells, insect cells or yeast cells.
• the host cells of the invention are preferably characterized by the fact that the introduced nucleic acid molecule of the invention either is heterologous with regard to the transformed cell, i.e. that it does not naturally occur in these cells, or is localized at a place in the genome different from that of the corresponding naturally occurring sequence.
• a further embodiment of the invention relates to isolated proteins exhibiting biological properties of the metastasis- associated antigen C4.4A and being encoded by the nucleic acid molecules of the invention, as well as to methods for their production, whereby, e.g, a host cell of the invention is cultivated under conditions allowing the synthesis of the protein and the protein is subsequently isolated from the cultivated cells and/or the culture medium. Isolation and purification of the recombinantly produced proteins may be carried out by conventional means including preparative chromatography and affinity and immunological separations involving affinity chromatography with monoclonal or polyclonal antibodies, e.g. with the antibody C4.4.
• isolated protein includes proteins substantially free of other proteins, nucleic acids, lipids, carbohydrates or other materials with which it is naturally associated. Such proteins however not only comprise recombinantly produced proteins but include isolated naturally occurring proteins, synthetically produced proteins, or proteins produced by a combination of these methods. Means for preparing such proteins are well understood in the art.
• the C4.4A proteins are preferably in a substantially purified form. A recombinantly produced version of C4.4A protein, including the secreted protein, can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988) .
• the invention relates to nucleic acid molecules specifically hybridizing to transcripts of the nucleic acid molecules of the invention.
• nucleic acid molecules can be used, for example, as probes in the diagnostic assay and/or kit described below and, preferably, are oligonucleotides having a length of at least 10, in particular of at least 15 and particularly preferred of at least 50 nucleotides.
• the nucleic acid molecules and oligonucleotides of the invention can also be used, for example, as primers for a PCR reaction.
• the present invention relates to an antisense RNA sequence characterized in that it is complementary to an mRNA transcribed from the nucleic acid molecule of the present invention or a part thereof and can selectively bind to said mRNA, said sequence being capable of inhibiting the synthesis of the protein encoded by said nucleic acid molecules, and a ribozyme characterized in that it is complementary to an mRNA transcribed from the nucleic acid molecule of the present invention or a part thereof and can selectively bind to and cleave said mRNA, thus inhibiting the synthesis of the proteins encoded by said nucleic acid molecules.
• Ribozymes which are composed of a single RNA chain are RNA enzymes, i.e.
• catalytic RNAs which can intermolecularly cleave a target RNA, for example the mRNA transcribed from the C4.4A gene. It is now possible to construct ribozymes which are able to cleave the target RNA at a specific site by following the strategies described in the literature, (see, e.g., Tanner et al . , in: Antisense Research and Applications, CRC Press Inc. (1993), 415-426). The two main requirements for such ribozymes are the catalytic domain and regions which are complementary to the target RNA and which allow them to bind to its substrate, which is a prerequisite for cleavage.
• Said complementary sequences are useful for repression of C4.4A expression, e.g. in the case of the treatment of a cell proliferative disorder associated with metastasizing tumors.
• the antisense RNA and ribozyme of the invention are complementary to the coding region of the C4.4A mRNA, e.g. to the 5' part of the coding region.
• the person skilled in the art provided with the sequences of the nucleic acid molecules of the present invention will be in a position to produce and utilize the above described antisense RNAs or ribozymes.
• the region of the antisense RNA and ribozyme, respectively, which shows complementarity to the mRNA transcribed from the nucleic acid molecules of the present invention preferably has a length of at least 10, in particular of at least 15 and particularly preferred of at least 50 nucleotides.
• the present invention relates to C4.4A inhibitors which fulfill a similar purpose as the antisense RNAs or ribozymes mentioned above, i.e. reduction or elimination of biologically active C4.4A molecules.
• Such inhibitors can be, for instance, structural analogues of the C4.4A protein that act as antagonists.
• inhibitors comprise molecules identified by the use of the recombinantly produced C4.4A, e.g. the recombinantly produced C4.4A can be used to screen for and identify C4.4A inhibitors, for example, by exploiting the capability of potential inhibitors to bind to C4.4A under appropriate conditions.
• the inhibitors can, for example, be identified by preparing a test mixture wherein the inhibtor candidate is incubated with C4.4A under appropriate conditions that allow C4.4A to be in a native conformation.
• Such an in vitro test system can be established according to methods well known in the art.
• Inhibitors of C4.4A can be identified, for example, by first screening for either synthetic or naturally occuring molecules that bind to the recombinantly produced C4.4A and then, in a second step, by testing those selected molecules in cellular assays for inhibition of CIITA, as reflected by inhibition of at least one of the biological activities of C4.4A as described in the Examples, below.
• Such screening for molecules that bind C4.4A could easily performed on a large scale, e.g. by screening candidate molecules from libraries of synthetic and/or natural molecules.
• Such an inhibitor is, e.g., a synthetic organic chemical, a natural fermentation product, a substance extracted from a microorganism, plant or animal, or a peptid
• the present invention also provides a method for detecting a cell proliferative disorder associated with a metastasizing tumor which comprises contacting a sample suspected to contain C4.4A or the C4.4A encoding mRNA with a reagent which reacts with C4.4A or the C4.4A encoding mRNA and detecting C4.4A or the C4.4A encoding mRNA.
• the reagent is typically a nucleic acid probe or a primer for PCR.
• the person skilled in the art is in a position to design suitable nucleic acids probes based on the information as regards the nucleotide sequence of C4.4A as depicted in Figure 1 and 2, respectively.
• the reagent is typically an antibody probe.
• antibody preferably, relates to antibodies which consist essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monclonal antibody preparations .
• Monoclonal antibodies are made from an antigen containing fragments of the C4.4A protein of the invention by methods well known to those skilled in the art (see, e.g., K ⁇ hler et al . , Nature 256 (1975), 495).
• antibody As used herein, the term "antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to protein. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody. (Wahl et al . , J. Nucl . Med. 24:316-325 (1983).) Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, and humanized antibodies.
• the target cellular component i.e. C4.4A or a C4.4A encoding mRNA, e.g., in biological fluids or tissues
• C4.4A or a C4.4A encoding mRNA may be detected directly in situ or it may be isolated from other cell components by common methods known to those skilled in the art before contacting with a probe.
• Detection methods include Northern blot analysis, RNase protection, in situ methods, PCR, LCR, immunoassays and other detection assays that are known to those skilled in the art.
• tissue samples include tissue of heart, renal, brain, colon, breast, urogenital, uterine, hematopoietic, prostate, thymus, lung, testis, pancreas and ovarian.
• a preferred tissue sample of the present invention is pancreas.
• the probes can be detectably labeled, for example, with a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, or an enzyme .
• C4.4A expression in tissues can be studied with classical immunohistological methods (Jalkanen et al . , J. Cell. Biol . 101 (1985), 976-985; Jalkanen et al . , J. Cell. Biol. 105 (1987), 3087-3096).
• Other antibody based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA) .
• Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine ( 125 I, 121 I) , carbon
• C4.4A can also be detected in vivo by imaging.
• Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR.
• suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
• Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
• a protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety such as a radioisotope (for example, 131 I, 112 In, 99 mTc) , a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally , subcutaneously, or intraperitoneally) into the mammal.
• a radioisotope for example, 131 I, 112 In, 99 mTc
• a radio-opaque substance for example, parenterally , subcutaneously, or intraperitoneally
• the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of "mTc .
• the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein.
• In vivo tumor imaging is described in S.W. Burchiel et al. , " Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds . , Masson Publishing Inc. (1982)).
• C4.4A encoding mRNA is normal or increased, thus indicative for the presence of a metastasizing tumor, the measured concentration is compared with the concentration in a normal tissue .
• the present invention also relates to a method for preventing, treating, or ameliorating a cell proliferative disorder associated with a metastasizing tumor which comprises administering to a mammalian subject a therapeutically effective amount of a reagent which decreases or inhibits C4.4A expression or the activity of C4.4A.
• a reagent which decreases or inhibits C4.4A expression or the activity of C4.4A.
• examples of such reagents are the above described antisense RNAs, ribozymes or inhibitors, e.g. C4.4A specific antibodies.
• administration of an antibody directed to C4.4A can bind and reduce overproduction of the protein.
• these reagents are preferably combined with suitable pharmaceutical carriers.
• suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emuslions, various types of wetting agents, sterile solutions etc..
• Such carriers can be formulated by conventional methods and can be administered to the subject at a suitabel dose.
• Administration of the suitable compositions may be effected by different ways, e.g. by intravenous, intraperetoneal, subcutaneous, intramuscular, topical or intrader al administration.
• the route of administration depends on the nature of the metastasizing tumor and the kind of compound contained in the pharmaceutical composition.
• the dosage regimen will be determined by the attending physician and other clinical factors.
• dosages for any one patient depends on many factors, including the patient's size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind of the metastasizing tumor, general health and other drugs being administered concurrently.
• the delivery of the antisense RNAs or ribozymes of the invention can be achieved by direct application or, preferably, by using a recombinant expression vector such as a chimeric virus containing these compounds or a colloidal dispersion system.
• a recombinant expression vector such as a chimeric virus containing these compounds or a colloidal dispersion system.
• the above nucleic acids can be administered directly to the target site, e.g., by ballistic delivery, as a colloidal dispersion system or by catheter to a site in artery.
• the colloidal dispersion systems which can be used for delivery of the above nucleic acids include macromolecule complexes, nanocapsules, microspheres , beads and lipid-based systems including oil-in-water emulsions, (mixed) micelles, liposomes and lipoplexes.
• the preferred colloidal system is a liposome.
• the composition of the liposome is usually a combination of phospholipids and steroids, especially cholesterol. The skilled person is in a position to select such liposomes which are suitable for the delivery of the desired nucleic acid molecule.
• Organ-specific or cell-specific liposomes can be used in order to achieve delivery only to the desired metastasizing tumor.
• the targeting of liposomes can be carried out by the person skilled in the art by applying commonly known methods. This targeting includes passive targeting (utilizing the natural tendency of the liposomes to distribute to cells of the RES in organs which contain sinusoidal capillaries) or active targeting (for example by coupling the liposome to a specific ligand, e.g., an antibody, a receptor, sugar, glycolipid, protein etc., by well known methods).
• a specific ligand e.g., an antibody, a receptor, sugar, glycolipid, protein etc.
• monoclonal antibodies are preferably used to target liposomes to specific tumors via specific cell- surface ligands.
• Preferred recombinant vectors useful for gene therapy are viral vectors, e.g. adenovirus, herpes virus, vaccinia, or, more preferably, an RNA virus such as a retrovirus.
• the retroviral vector is a derivative of a murine or avian retrovirus .
• retroviral vectors which can be used in the present invention are: Moloney murine leukemia virus (MoMuLV) , Harvey murine sarcoma virus (HaMuSV) , murine mammary tumor virus (MuMTV) and Rous sarcoma virus
• a non-human primate retroviral vector is employed, such as the gibbon ape leukemia virus (GaLV) , providing a broader host range compared to murine vectors. Since recombinant retroviruses are defective, assistance is required in order to produce infectious particles. Such assistance can be provided, e.g., by using helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. Suitable helper cell lines are well known to those skilled in the art. Said vectors can additionally contain a gene encoding a selectable marker so that the transduced cells can be identified.
• GaLV gibbon ape leukemia virus
• the retroviral vectors can be modified in such a way that they become target specific. This can be achieved, e.g., by inserting a polynucleotid encoding a sugar, a glycolipid, or a protein, preferably an antibody.
• a polynucleotid encoding a sugar, a glycolipid, or a protein, preferably an antibody.
• Those skilled in the art know additional methods for generating target specific vectors. Further suitable vectors and methods for in vitro- or in vivo- gene therapy are described in the literature and are known to the persons skilled in the art; see, e.g., WO 94/29469 or WO 97/00957.
• the nucleic acids encoding e.g. an antisense RNA or ribozyme can also be operably linked to a tissue specific promotor and used for gene therapy.
• tissue specific promotor e.g. an antisense RNA or ribozyme
• Such promotors are well known to those skilled in the art (see e.g. Zimmermann et al , 1994, Neuron 12, 11-24; Vidal et al . 1990, EMBO J. 9, 833-840; Mayford et al . , 1995, Cell 81, 891-904; Pinkert et al . , 1987, Genes & Dev. 1, 268- 76) .
• kits are also provided by the present invention. Such kits are useful for the detection of a target cellular component, which is C4.4A or C4.4A encoding mRNA, wherein the presence or an increased concentration of C4.4A or C4.4A encoding mRNA is indicative for a cell proliferative disorder associated with a metastasizing tumor, said kit comprising a probe for detection of C4.4A or C4.4A encoding mRNA.
• the probe can be detectably labeled.
• Such probe may be an antibody or oligonucleotide specific for C4.4A or C4.4A encoding mRNA.
• said kit contains a C4.4A specific antibody and allows said diagnosis by ELISA and contains the antibody bound to a solid support, for example, a polystyrene microtiter dish or nitrocellulose paper, using techniques known in the art.
• said kits are based on a RIA and contain said antibody marked with a radioactive isotope.
• the C4.4A-antibody is labelled with enzymes, fluorescent compounds, luminescent compounds, ferromagnetic probes or radioactive compounds.
• the kit of the invention may comprise one or more containers filled with, for example, one or more probes of the invention.
• container (s) of the kit can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
• BDX rats were obtained from Charles River, Sulzfeld, Germany. They were kept under SPF conditions, fed with conventional diet and water at libitum. They were used for experiments at the age of 8 to 10 weeks.
• BSp73AS-C4.4A (BSp73A transfected with cDNA coding for the C4.4A molecules) and BSp73AS-mock (BS73AS transfected with the pCDNA3-vector) are described below. All these tumor lines were derived from the BDX rat strain. The tumor lines regressor (RG) and progressor (PROG) , a metastasizing colon carcinoma of the BDIX strain has been kindly provided by F.Martin (Reisser et al . , Int. J. Cancer 53. (1993), 651-656). The adherently growing tumor lines were cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS) .
• FCS fetal calf serum
• Confluent cultures were either trypsinized (BSp73ASML, PROG and RG) or treated with EDTA (BSp73AS, BSp6S, BSp3A) and split.
• COS-7 cells were obtained from the DSMZ. They were cultured in DMEM supplemented with 10% fetal calf serum.
• mAB C4.4 mouse IgGl
• an isotype matched control antibody, 3-9, binding a Ga-chelate complex have been described previously (Matzku et al . , Cancer Res. 49 . (1989), 1294-1299; Zoller et al . , J.Nucl.Med. 33 (1992), 1366-1372).
• a rabbit anti-rat uPAR was obtained from American Diagnostics. Polyclonal rabbit anti-mouse IgGl and fluoresecence dye labeled derivatives as well as an FITC labelled anti-rat IgG were obtained from Southern Biotechnology (Birmingham, AL) .
• cDNA library and selection of clones coding for the rat surface molecule C4.4A mRNA was prepared from the colon carcinoma cell line RG and oligo(dT) . Unprimed cDNA was prepared using the Librarian Kit (Invitrogen, San Diego) . The cDNA was inserted unidirectional into the mammalian expression vector pcDNA3 after ligation of BstXI adaptors and cleavage with Notl . The ligated DNA was transformed into E.coli TOP10P. After cultivation, isolation of the plasmid DNA and plating of aliquots, the size of the library was estimated to about 2 x 10 7 clones.
• the plasmid DNA/RNA mixture was digested with RNase A (20 ⁇ g/ml) and Proteinase K (50 ⁇ g/ml), followed by a phenol-chloroform extraction to remove residual proteins. The remaining plasmid DNA was precipitated and used for the transformation of E.coli by electroporation. The procedure was repeated three times. Thereafter the plasmid DNA was isolated from single bacterial colonies and was used for DEAE transfection of COS-7 cells. After 3 days cells were analysed for antigen expression by FACS. The cDNA of positive clones was sequenced according to the method of Sanger.
• the tumor lines BSp73AS, BSp6S and BSp3A were harvested during growth in log phase.
• Cells (1 x 10 7 /ml) were resuspended in RPMI 1640 containing 10% FCS and were mixed with 15 ⁇ g of the pcDNA3-C4.4A plasmid in a steril cuvette. Electroporation was performed after 15 min. incubation at room temperature at 260 V and 1050 ⁇ F using a Bio-Rad Genepulser with capacitance extender.
• Transfected cells were selected by growth in RPMI 1640 containing 10% FCS and 500 ⁇ g/ml G418. G418 resistant cells were analysed for C4.4A expression by flowcytometry and were cloned.
• cDNA was synthesized and subjected to PCR amplification using 2 ⁇ g total RNA and 1 ⁇ g oligo(dT) 15 for synthesis of the first strand and C4.4A-specific primers for the amplification.
• the following C4.4A specific oligonucleotides were used: 5 ' TGCTACAGCTGCGTGCAA and 3 ' TTGGAACTGCGGATGCTG .
• Amplification was performed at 59°C with 5' and 3' oligonucleotides for 35 cycles.
• Amplification of GAPDH was performed accordingly at 53°C.
• PCR products were analysed on a 1% agarose gel.
• SDS-PAGE was performed under non-reducing conditions using 8 or 10% gels according to the method of Laemmli (Nature 227 (1970) , 680-685) . Gels were transferred to PVDF membranes (Millipore) by the method of Towbin et al . (Proc.Natl.Acad.Sci. USA 76 (1979), 4350-4354). Membranes were blocked by incubation in PBS containing 5% (w/v) non-fat dry milk powder and 0,1% (v/v) Tween 20® for 1 h at room temperature.
• membranes were washed four times with PBS/0, 1% Tween 20®, incubated for 1 h with goat anti-mouse IgGl- horseradish peroxidase (HRPO) (1:5000 in PBS/0,1% Tween 20®) and washed four times with PBS/0, 1% Tween 20®.
• HRPO horseradish peroxidase
• Anchorage, glycosyla ion and solubility N- and O-glycosylation were inhibited by plating 2 x 10 5 cells (six well plate) and growing the cells in RPMI 1640, 10% FCS containing either 2,5 ⁇ g/ml Tunicamycin or 2 mM phenyl-alpha- GalNac. Thereafter cells were harvested, suspended in 300 ⁇ l SDS sample buffer and analysed by Western blot.
• HA extracellular matrix
• LA laminin
• fibronectin fibronectin
• vitronectin components of the extracellular matrix, except for HA were coated at 10 ⁇ g/ml; HA was coated at a concentration of 100 ⁇ g/ml.
• the protease inhibitor aprotinin (100 ⁇ g/ml) was added to exclude laminin degradation.
• anti-C4.4 (10 ⁇ g/ml) was added to the culture medium.
• Cells were incubated for 30 min. to 2 h and were washed repeatedly.
• the adherent cells were lysed by 2% SDS, transferred into counting vials and counted in a ⁇ -counter. The percentage of adherent cells is shown.
• the migratory activity of C4.4A + cells was evaluated in a matrigel assay: Membranes with a pore size of 8 ⁇ m were coated with 50 ⁇ l matrigel (Sigma, St. Louis, MO, USA). The membranes were inserted into 24 well plates which contained medium without supplements. Tumor cells (1 x 10 4 ) were suspended in RPMI 1640 supplemented with 10% FCS and, where indicated, with 10 ⁇ g/ml C4.4 and were seeded on the matrigel. Plates were incubated for 48 h at 37°C. Thereafter cells at the bottom of the chamber were counted using an inverted microscope.
• C4.4A transfected cells Proliferation of C4.4A transfected cells was tested by [ 3 H] thymidine incorporation.
• Cells were seeded on control IgG or C4.4 coated plates. The supernatant contained either a control antibody or C4.4.
• [ 3 H] thymidine was added immediately or after 24 to 72 h. After 8 h of incubation with [ 3 H] thymidine cells were trypsinized and transferred to a ⁇ -counter to evaluate thymidine incorporation.
• BDX rats received 5 x 10 5 tumor cells, i.v. or i.f.p.. In the latter case the tumor and the draining lymph node were excised by amputation in the knee as soon as the primary tumor reached a diameter of 0.5 cm. Animals were observed for local tumor growth, weight loss, anemia and shortage of breathing. They were sacrified before reaching a moribund stage. Metastasis formation was controlled macroscopically and by histology.
• hematopoietic malignancies B ⁇ , BxPC-3, 8.18 (carcinoma of the pancreatic gland), KTCM-1M, KTCM-129, KTCM-140 (renal cell carcinoma) , MKN (stomach carcinoma) , Igrov (carcinoma of the ovary) , JAR (choriocarcinoma) , COLO-680 (esophagus carcinoma) ,
• Image filter No. 10 containing Soares 2NbHP8-9W normal 8 to 9 week placental library was screened with a 32 P labeled C4.4A cDNA probe of the rat.
• One of the labeled clones (IMAGEp9 8 Ao5562Q6) was sequenced using the chain termination method and compared with the rat sequence.
• FISH Fluorescence in situ hybridization
• a PAC Pl-derived artificial chromosome
• ICRF library No704, Resource Center Primary Data Base, German Human Genome Porject, Max Planck Institute for Molecular Genetics, Berlin Two positive clones (LLNLP704 F09964Q3 and LLNLP704 D03144Q19) were isolated and used for FISH analysis.
• PAC-DNA was labeled with DIG-11-dUTP (Boehringer, Mannheim) by nick translation. Suppression of repetitive sequences, denaturation, hybridization and fluorescence detection were performed according to routine procedures.
• Anti-DIG mouse IgG K (Boehringer, Mannheim) and Cy3-conjugated sheep anti-mouse IgG (Dianova) were used to detect Digoxigenin labeled probes. Chromosomes were counterstained with DAPI . Analysis was performed using a Zeiss Axiophot microscope. Images were collected and merged using a cooled CCD camera (KAF 1400, Photometries) and IPLab Spectrum software .
• RNA was prepared by the guanidine isothiocyanate/acid phenol method.
• cDNA was synthesized and subjected to RT-PCR amplification using 9 ⁇ g of total RNA and 25 pmol of random hexanucleotides for the first strand synthesis and the following human C4.4A specific oligonucleotides for the amplification (32 cycles at 60° C) : GCC CCA GCA GCC CCA TAA TAA A and CAC CCA CCC CAC GCT CCA AAG T.
• oligonucleotides AAC GAC ACC TTC CAC TTC and GCA CAG CCT CTT ACC ATA have been used.
• Amplification of GAPDH oligonucleotides: ACC ACA GTC CAT GCC ATC AC and TCC ACC ACC CTG TTG CTG TA was performed for 35 cycles at 56° C.
• RT-PCR products were analyzed on 1% or 2% agarose gels stained with ethidium bromide. The identity of the fragments was checked by sequencing.
• RNA total RNA from human tissue or tumor cells were loaded per lane on a denaturing agarose gel . After gel electrophoresis the RNA was transferred to a positively charged nylon membrane by vacuum transfer. After UV- crosslinking of nucleic acid with a Stratal inker , hybridization was done with radioactively labeled RT-PCR fragments of human C4.4A, uPAR or GAPDH with stringent washing. A second filter with human poly (A) -RNA was obtained from Clontech (multiple tissue northern blot, MTNTM) .
• melanoma lines were starved overnight in RPMI 1640 medium not containing fetal calf serum. Thereafter they were cultured for 24 hours in medium containing 10 % heat inactivated fetal calf serum or 10 % ABO serum which was or was not heat-inactivated for 30 min at 56°C.
• RNA-probe In order to generate a human C4.4A-specific RNA-probe we first performed RT-PCR with 5 ⁇ g RNA using the following primers: 5'- GCCCCAGCAGCCCCATAATAAA (hC4.4A 1037-1058) and 5'- CACCCACCCCACGCTCCAAAGT (hC4.4A 1483-1504). The resulting DNA product of 467 bp was gel-eluted and purified using the Qiaquick Gel Extraction Kit (Quiagen, Hilden, Germany) and subsequently cloned into the pCRII-Topo vector (Invitrogen, Groningen, The Netherlands). After sequence analysis, sense and antisense probes were generated by in vitro-transcription using T7 or Sp6 RNA-polymerase. The probe was digoxigenin
• RT-PCR mRNA was isolated with the Oligotex mRNA purification system (Quiagen, Hilden, Germany) using the manufacturers protocol. cDNA was synthesized using l ⁇ g of polyA + RNA, MuMLV reverse transcriptase and oligo dT . First strand cDNA was subjected to RT-PCR amplification. Using polyA + RNA-derived cDNA the following human C4.4A specific oligonucleotides have been used for the amplification (32 cycles at 55°C) : GCC CCA GCA GCC CCA TAA TAA A and CAC CCA CCC CAC GCT CCA AAG T.
• GAPDH oligonucleotides: GGT CGG AGT CAA CGG ATT TG and ATG AGC CCC AGC CTT CTC CAT
• the 4.4A-specific and the GAPDH-specific primers amplify a PCR fragment of 460bp and 400 bp, respectively.
• RT-PCR products were analyzed on 1 % agarose gels stained with ethidium bromide.
• 2 ⁇ l of cDNA was added to 18 ⁇ l PCR mix (LightCycler Fast Start DNA Master SYBR Green I kit, Roche Diagnostics) .
• SYBR Green intercalates between double-stranded DNA and a fluorescence signal is generated through a laser beam. Fluorescence emission is measured and continuously monitored during PCR. The fluorescence signal is plotted versus cross points which mark the cycle number when fluorescence becomes significantly different from baseline signal .
• COS-7 cells were repeatedly transfected with plasmid DNA of a cDNA library derived from the metastatic rat colon carcinoma line RG.
• C4.4A positive cells were selected by fluorescence staining and FACS sorting. After three extractions according to the method of Hirt 12 bacterial colonies were selected and cDNA of individual colonies was transfected into COS-7 cells. One clone was isolated which gave positive FACS staining with C4.4 mAB.
• Western blot analysis confirmed that the cells expressed a C4.4-reactive molecule of an estimated molecular weight of about 94 kDa, corresponding to the molecule expressed on RG, where the cDNA was derived from.
• the C4.4A molecule of the metastatic ASML line had a slightly higher molecular weight.
• a placental library was screened with a 32 P labeled C4.4A cDNA probe of the rat.
• One of the labeled clones was full length sequenced and compared with the rat sequence.
• the cDNA sequence and the deduced amino acid sequence are shown in Figure 2.
• the homology of human to rat C4.4A is 72.2% at the DNA level and 81.8% at the amino acid level ( Figure 7). This corresponds to the homology between rat C4.4A and rat uPAR (44,9% at the cDNA level and 46,9% on the amino acid level).
• the rat C4.4A molecule potentially spans 352 aa.
• the molecule has consensus sequences for seven potential N- glycosylation sites. Like the uPAR, it can be divided into three domains, where domains 1 and 2 show some homology to the uPAR of several species, while the third domain is unrelated. According to the aa sequence the C4.4A molecule has an isoelectric point of pH 7.04 and a theoretical MW of 36.96 kDa.
• Western blotting under non-reducing conditions revealed molecular weights of 98 kDa (ASML) and 94 kDa (PROG, AS-lBl, AS-2A2) ( Figure 4) .
• the apparanetly higher molecular weight as revealed by Western blotting after tunicamycin treatment may be a consequence of additional modifications of the GPI anchor (see below) , but ist not due to dimerization, because the MW of the molecule in immunoprecipitates after surface biotinylation is the same under reducing and non-reducing conditions.
• the C-terminal sequence of C4.4A suggests that the molecule may be phosphatidyl-inositol anchored. This suggestion was strengthened by the observation of a high degree of Triton X-100 insolubility of C4.4A, which is characteristic for GPI anchored molecules (Moller et al . , FEBS Lett. 301 (1992), 493-500).
• C4.4A The GPI anchorage of C4.4A was finally proven by treatment with phosphatidyl-inositol phospholipase C. As shown by Western blotting and by fluorescence staining, C4.4 was removed from the cell membrane after phosphatidyl-inositol phospholipase C treatment. Like in the rat, the homolgy between human C4.4A and uPAR is restricted to domains 1 and 2, whereas domain 3 shows no homology. Human C4.4A has 6 N-glycosylation sites. Like the rat C4.4A as well as the rat and human uPAR it contains a potential GPI-anchoring sequence.
• C4.4A gene like the uPAR gene, is located on chromosome 19ql3.1- ql3.2 ( Figure 8).
• C4.4A and uPAR are the only members of the Ly-6 superfamily (Rock et al . , Immmunol . Rev. Ill (1989), 195-224) which are composed of 3 domains, while all other members consist of 1 domain (Ploug and Ellis, FEBS Lett. 349 (1994), 163-168) .
• Rat C4.4A originally has been detected only on rat tumor lines, which metastasize via the lymphatic system. Thus, it was of special interest, whether C4.4A would be involved in the process of tumor progression.
• C4.4A would be involved in the process of tumor progression.
• the two C4.4A transfected BSp73AS lines, AS-lBl and AS-2A2 were inoculated intravenously and survival time as well as metastasis formation were compared to mock-transfected cells. It became apparent that the survival time of all three groups was comparable and that all rats developed lung metastasis.
• Phosphatidyl-inositol anchored molecules display a variety of functions (Moller, Blood Coagul . Fibrinolysis 4
• C4.4A + cells The transient adhesion of C4.4A + cells to laminin strengthened the hypothesis that C4.4 may enable cells for degradation of elements of the extracellular matrix. Indeed when constitutively C4.4A + cells BSp73ASML cells were layered on matrigel coated transwell plates, 66% migrated through the matrix within 24 h. In the presence of C4.4 (lO ⁇ g/ml) migration through the matrix was nearly completely inhibited. However, only a minority of cells transfected with C4.4A + cDNA penetrated through the matrigel.
• C4.4A transfected BSp73AS cells could penetrate the matrigel.
• C4.4A + line BSp73ASML uPA or a corresponding molecule might bind to C4.4A, thus initiating the process of matrix degradation.
• the transfected lines can recruit in vivo uPA or a homologous molecule from host cells, these experiments could provide a hint, why C4.4A + tumor cells metastasize without capsule formation and by which mechanism C4.4A facilitates metastasis formation.
• hC4.4A expression of hC4.4A in normal tissue was evaluated by Northern blots using a filter containing poly (A) -RNA from brain, placenta, lung, liver, skeletal muscle, kidney and pancreas obtained by Clontech ( Figure 9a) and by RT-PCR of human cDNA of skin, oesophagus, thymus , colon, spleen, kidney, lung, brain, liver, stomach and peripheral blood leukocytes
• hC4.4A While the expression of hC4.4A on non-transformed tissue was very restricted, hC4.4A mRNA was found in 56% of tumor lines. This has been evaluated by RT-PCR ( Figure 10) and confirmed by Northern blots using filters containing 10 ⁇ g of total RNA per lane. 9 colon carcinoma, 20 malignant melanoma, 5 mammary carcinoma, 5 malignancies of the hematopoietic system, 3 pancreatic adenocarcinoma, 3 renal cell carcinoma, 1 or 2 tumor cell lines of liver, chorion, stomach, esophagus, lung, cervix, ovary and 1 fibrosarcoma were tested. Human C4.4A was detected in 2 of 5 tested malignancies of the hematopoietic system. It was also found in 100% of malignant melanoma, 22% of colon carcinoma lines and 20% of mamma carcinoma lines
• the present invention identifies cDNAs encoding rat and human C4.4A, respectively.
• the rat C4.4A cDNA has been isolated from a metastasizing tumor line, i.e. is metastasis- associated expressed.
• the rat and human C4.4A is hardly expressed in normal tissues of the adult organism.
• C4.4A did not alter the metastasic potential of tumor cells, but influenced the manner of host tissue invasion: Metastasis formation of C4.4A transfected lines was either miliary or at least without any form of encapsulation.
• the GPI anchored C4.4A molecule displays structurally and functionally similarities to the uPAR and facilitates embedding of metastasizing tumor cells likely by degradation of the extracellular matrix.
• the results presented above suggest that an interference with metastasis formation by blockade of the C4.4A molecule is achievable.
• the C4.4A molecule could as well function as marker for metastasizing tumors, propably at an early stage of the disease, thus allowing a therapeutical treatment at an early stage and, as a consequence, increasing the prospects of effective curing.
• the C4.4A molecule may also be useful for evaluating whether a metastasizing tumor is resistant to particular therapeutic regimens.
• hC4.4A expression of hC4.4A was evaluated by RT-PCR and ISH in melanocytes, unaltered skin, 7 nevi, 10 primary malignant melanoma, 8 lymph node and 7 skin metastasis of malignant melanoma (Table II) .
• human skin weakly expressed hC4.4A, the expression being restricted to the stratum basale.
• melanocytes nor nevi expressed hC4.4A.
• primary malignant melanoma the picture was not uniform.
• RT-PCR no signal was obtained with 3 out of 10 tumors, weak signals were seen with 5 and strong signals with 2 samples. All skin and lymph node metastases expressed hC4.4A, the signals being in most instances stronger than those of primary tumors.
• hC4.4A is a differentiation marker of cells of the melanocytic lineage. Furthermore, hC4.4A is not expressed constitutively on malignant melanoma cells, but its expression appears to be upregulated during tumor progession.
• hC4.4A Activational state dependent expression of hC4.4A Because of the higher expression level of hC4.4 on metastases of malignant melanoma, it became plausible to speculate that the acivational state of tumor cells may have bearing on hC4.4A expression.
• malignant melanoma cell lines were starved overnight, i.e. were cultured in the absence of fetal calf serum. Thereafter they were cultured in the presence of human serum, which had not been heat inactivated, and expression of hC4.4A was evaluated by quantitative PCR. Transcription of hC4.4A became upregulated by all 4 tested lines, the increase spanning a range from 2.3- to 8.2-fold. Importantly, when the cells were cultured in the presence of heat inactivated human serum, no such increase was observed, i.e. expression was in the same range or below the one of cells cultured in the presence of heat inactivated fetal calf serum.
• rat C4.4A facilitates matrix degradation and interferes with adhesion to laminin.
• the finding that expression of hC4.4A is upregulated on metastasizing melanoma cells could well be in line with functional activity of hC4.4A in matrix degradation and is reminiscent of uPAR expression on colon carcinoma which correlates with the metastastic capacity.
• Activation of transcription is another phenomenon shared with the uPAR gene, transcription of which has been described to be regulated by nerve growth factor, epidermal growth factor, serum factor VII and Vila as well as by TGF- beta 1.
• Our data indicate a very restricted expression in non- transformed cells and a high expression on metastases.
• hC4.4A will possibly be important as a prognostic indicator and a therapeutic target.

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