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WO1998023730A9 - Composes et methodes de traitement du cancer - Google Patents

Composes et methodes de traitement du cancer

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Publication number
WO1998023730A9
WO1998023730A9 PCT/US1997/022087 US9722087W WO9823730A9 WO 1998023730 A9 WO1998023730 A9 WO 1998023730A9 US 9722087 W US9722087 W US 9722087W WO 9823730 A9 WO9823730 A9 WO 9823730A9
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cells
wnt
gene
tumor
tumor cells
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  • the present invention relates to compounds and methods for treating cancer, and in particular, compositions and methods for transfecting tumor cells under conditions such that tumor growth is suppressed.
  • total cell kill implies that total excision of a tumor is necessary for a surgical approach, and complete destruction of all cancer cells is needed in a radiation approach, if one is to achieve a cure. In practice this is rarely possible; indeed, where there are metastases, it is impossible.
  • chemotherapy simply means the treatment of disease with chemical substances.
  • This target specificity is sought in all types of chemotherapeutics, including anticancer agents.
  • specificity has been the major problem with anticancer agents. In ⁇ the case of anticancer agents, the drug needs to distinguish between host cells that are cancerous and host cells that are not cancerous. The vast bulk of anticancer drugs are indiscriminate at this level.
  • anticancer agents typically have negative hematological effects (e.g., cessation of mitosis and disintegration of formed elements in marrow and lymphoid tissues), and immunosuppressive action (e.g., depressed cell counts), as well as a severe impact on epithelial tissues (e.g., intestinal mucosa), reproductive tissues (e.g., impairment of spermatogenesis), and the nervous system.
  • negative hematological effects e.g., cessation of mitosis and disintegration of formed elements in marrow and lymphoid tissues
  • immunosuppressive action e.g., depressed cell counts
  • epithelial tissues e.g., intestinal mucosa
  • reproductive tissues e.g., impairment of spermatogenesis
  • the invention generally relates to the treatment of cancer, and, more specifically, to the treatment of cancer, including metastases, without radiation or toxic chemotherapeutic agents.
  • the invention involves compositions and methods for transfecting tumor cells under conditions such that tumor growth is suppressed, and in particular, transfection of nucleic acid comprising the wnt-5a gene.
  • Wnt-gene family members are thought to play an important role in cell growth and differentiation. When normal wnt-gene expression is disrupted there is the potential for cell transformation.
  • the present invention contemplates the use of antisense wnt-5a as well as expressing of the wnt-5a gene product in tumor cells such that the tumorogenic phenotype is transformed into a non-tumorogenic phenotype.
  • the present invention contemplates a method, comprising: a) providing i) tumor cells and ii) nucleic acid encoding at least a portion of the wnt-5a gene, and b) introducing said nucleic acid into said tumor cells.
  • said nucleic acid encodes the entire wnt-5a gene and said introducing comprises transfection under conditions such that a functional wnt-5a gene product is expressed by said tumor cells.
  • the present invention contemplates a composition comprising tumor cells containing a vector comprising nucleic acid which encodes at least a portion of the wnt-5a gene.
  • Figure 1A shows Northern analysis of two different clones of tumor cells stably expressing wnt-5a.
  • Figure IB is a graph showing growth curves of the stably expressing cells.
  • Figure 2 is a graph showing tumor volume measurements for transfected tumor cells (and controls) in vivo.
  • Figure 3 shows the telomerase activity of transfected tumor cells (and controls) as measured by the TRAP assay.
  • the term “gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or precursor thereof.
  • the polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity is retained.
  • the term “gene” encompasses both cDNA and genomic forms of a given gene. In some embodiments (such as antisense), portions of the gene are contemplated that, while too small to encode a functional protein, are of sufficient size to hybridize to complementary nucleic acid.
  • wild-type refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally occurring source.
  • a wild- type gene is that which is most frequently observed in a population and is thus arbitrarily designated the "normal” or “wild-type” form of the gene.
  • the term “modified” or “mutant” refers to a gene or gene product which displays modifications in sequence and or functional properties (/ ' . e. , altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
  • oligonucleotide as used herein is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, usually more than three (3), and typically more than ten (10) and up to one hundred (100) or more (although preferably between twenty and thirty). The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide.
  • the oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof.
  • the present invention contemplates an oligonucleotide capable of hybridizing to a portion of the wnt-5a gene for use in antisense therapy.
  • regulatory element refers to a genetic element which controls some aspect of the expression of nucleic acid sequences.
  • a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region.
  • Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc.
  • Transcriptional control signals in eucaryotes comprise "promoter” and “enhancer” elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription [Maniatis, T. et al, Science
  • Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in procaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types [for review see Voss, S.D. et al, Trends Biochem. Sci., 11 :287 (1986) and Maniatis, T. et al, supra (1987)].
  • expression vector and "recombinant DNA vector” as used herein refer to DNA sequences containing a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g. mammal).
  • DNA sequences necessary for expression in procaryotes include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences.
  • Eukaryotic cells are known to utilize promoters, polyadenlyation signals and enhancers.
  • operable combination refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced.
  • the term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
  • genetic cassette refers to a fragment or segment of DNA containing a particular grouping of genetic elements. The cassette can be removed and inserted into a vector or plasmid as a single unit.
  • a plasmid backbone refers to a piece of DNA containing at least plasmid origin of replication and a selectable marker gene (e.g., an antibiotic resistance gene) which allows for selection of bacterial hosts containing the plasmid; the plasmid backbone may also include a polylinker region to facilitate-the insertion of genetic elements within the plasmid.
  • a selectable marker gene e.g., an antibiotic resistance gene
  • an end of an oligonucleotide is referred to as the "5' end” if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the "3' end” if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring.
  • a nucleic acid sequence even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends.
  • primer refers to an oligonucleotide which is capable of acting as a point of initiation of synthesis when placed under conditions in which primer extension is initiated.
  • An oligonucleotide “primer” may occur naturally, as in a purified restriction digest or may be produced synthetically.
  • a primer is selected to be "substantially" complementary to a strand of specific sequence of the template.
  • a primer must be sufficiently complementary to hybridize with a template strand for primer elongation to occur.
  • a primer sequence need not reflect the exact sequence of the template.
  • a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being substantially complementary to the strand.
  • Non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize and thereby form a template primer complex for synthesis of the extension product of the primer.
  • Hybridization methods involve the annealing of a complementary sequence to the target nucleic acid (the sequence to be detected). The ability of two polymers of nucleic acid containing complementary sequences to find each other and anneal through base pairing interaction is a well-recognized phenomenon.
  • the initial observations of the "hybridization” process by Marmur and Lane, Proc. Natl Acad. Sci. USA 46:453 (1960) and Doty et al, Proc. Nad. Acad. Sci. USA 46:461 (1960) have been followed by the refinement of this process into an essential tool of modern biology.
  • nucleic acid sequence refers to -an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3' end of the other, is in "antiparallel association.”
  • Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention and include, for example, inosine and 7-deazaguanine.
  • duplex stability need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases.
  • Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs.
  • T m melting temperature
  • the T m of a particular nucleic acid duplex under specified conditions is the temperature at which on average half of the base pairs have disassociated.
  • the equation for calculating the T m of nucleic acids is well known in the art.
  • probe refers to a labeled oligonucleotide which forms a duplex structure with a sequence in another nucleic acid, due to complementarity of at least one sequence in the probe with a sequence in the other nucleic acid.
  • label refers to any atom or molecule which can be used to provide a detectable (preferably quantifiable) signal, and which can be attached to a nucleic acid or protein. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like.
  • transfection refers to the introduction of DNA (and more typically, foreign DNA) into eukaryotic cells.
  • the present invention specifically contemplates introducing nucleic acid into tumor cells, and more particularly, transfecting tumor cells with a gene of interest.
  • stable transfection or “stably transfected” refers to the introduction and integration of foreign DNA into the genome of the transfected cell.
  • stable transfectant refers to a cell which has stably integrated foreign DNA into the genomic DNA.
  • the term "gene of interest” refers to a gene inserted into a vector or plasmid whose expression is desired in a host cell.
  • the present invention contemplates that the wnt-5a gene is a genes of interest include having therapeutic value.
  • nucleic acid molecule encoding As used herein, the terms “nucleic acid molecule encoding,” “DNA sequence encoding,” and “DNA encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.
  • the present invention relates to compounds and methods for treating cancer, and in particular, compositions and methods for transfecting tumor cells under conditions such that tumor growth is suppressed.
  • chromosome 3 has a particularly high frequency of deletion or rearrangement in human cancers including small cell lung carcinoma, oral squamous cell carcinoma, cervical carcinoma, breast carcinoma, renal cell carcinoma, and uroepithelial cell carcinoma.
  • RFLP restriction fragment length polymorphism
  • 3p tumor suppressor gene(s) Although the precise location of the 3p tumor suppressor gene(s) is not known, cytogenetic analysis suggests that the region 3pl l-3p25 likely carries one or more suppressor genes. More specifically, Yamakawa et al. mapped one suppressor gene to 3p 13-p 14.2 and another distal to 3 ⁇ 2 1.3 in renal cell carcinoma. Yamakawa et al. , "A detailed mapping of the short arm of chromosome 3 in sporadic renal cell carcinoma," Cancer Res. 51 : 4707-4711
  • Wnt-genes Human wnt-5a has been cloned and mapped to human chromosome 3p 14-p21.
  • Wnt genes consist of a family of locally acting growth factor-like molecules which are involved in pattern formation, morphogenesis, and cell growth and differentiation.
  • Wntl, wnt-2 and wnt-3 are known to be activated by mouse mammary tumor virus (MMTV) proviral insertional mutagenesis in certain mouse mammary tumors. It is not known how some wit-gene family members are involved in cell transformation and tumorigenesis, but it likely involves disruption of normal spatio-temporal wnt gene expression.
  • MMTV mouse mammary tumor virus
  • the invention involves compositions and methods for transfecting tumor cells under conditions such that tumor growth is suppressed, and in particular, transfection of nucleic acid comprising the wnt-5a gene. It is not intended that the present invention be limited by the method of transfection.
  • the present invention contemplates that transfection may be accomplished by a variety of means known to the art including (but not limited to) calcium phosphate-DNA co-precipitation, DEAE-dextran- mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.
  • the present invention contemplates transfection in vitro and in vivo.
  • the present invention contemplates tumors having deletions for chromosome 3p.
  • a variety of tumor types are available for in vitro transfection, including but not limited to breast cancer cells, prostate cancer cells, and renal cancer cells. While the cancer cells of animals in general may be transfected, the present invention specifically contemplates the transfection of human cancer cells.
  • the present invention contemplates an SV40-immortalized human uroepithelial cell (SV-HUC- 1) cell line which showed nonrandom losses of chromosome 3p in association with tumorigenic transformation to high grade cancers. See Klingelhutz et al., "Allelic 3p deletions in high-grade carcinomas after transformation in vitro of human uroepithelial cells," Genes, Chrom. and Cancer 3: 346-357 (1991)
  • Transfection (whether stable or transient) can be achieved by use of vectors containing the wnt-5a gene, or functional variants thereof.
  • the nucleic acid sequence of wnt-5a cDNA and the amino acid sequence of the wnt-5a gene product are known (GENBANK Accession No. L20861).
  • Functional variants are those genes containing variations in sequence (i.e. non-wild type or mutant sequences) that do not change the function of the nucleic acid (such as in antisense, discussed below) or the function of the resulting wnt-5a gene product (i.e. the tumor suppressing nature of the gene product).
  • Such variants can be readily tested for functionality using the in vitro methods of the present invention described below (see Experimental).
  • vectors can be used, including but not limited to RSV expression vectors (commercially available from Invitrogen, San Diego, CA).
  • RSV expression vectors commercially available from Invitrogen, San Diego, CA.
  • transfection schemes with RSV expression vectors are known in the art. See e.g. U.S. Patent No.
  • the present invention contemplates transfection in vitro and in vivo.
  • a variety of strategies are contemplated including but not limited to systemic exposure to a vector comprising nucleic acid encoding at least a portion of the wnt-5a gene.
  • Wu et al U.S. Patent 5,166,320 (hereby incorporated by reference), discloses tissue-specific delivery of DNA using a conjugate of a polynucleic acid binding agent (such as polylysine, polyarginine, polyornithine, histone, avidin, or protamine) and a tissue receptor-specific protein ligand.
  • a polynucleic acid binding agent such as polylysine, polyarginine, polyornithine, histone, avidin, or protamine
  • asialoglycoprotein (galactose-terminal) ligands for targeting liver cells, using the above-described expression vectors.
  • Wagner et al Proc. Natl Acad.
  • the present invention contemplates oligonucleotides serving as antisense strands to the wnt-5a gene.
  • antisense strand is used in reference to a nucleic acid strand that is complementary to the "sense” strand.
  • the designation (-) i.e., "negative” is sometimes used in reference to the antisense strand, with the designation (+) sometimes used in reference to the sense (i.e. "positive”) strand.
  • the oligonucleotides of the present invention are RNA sequences which are complementary to a specific wnt-5a RNA sequence (e.g., wnt-5a mRNA).
  • Antisense RNA may be produced by any method, including synthesis by splicing the gene(s) of interest in a reverse orientation to a viral promoter which permits the synthesis of a coding strand. Once introduced, this transcribed strand combines with the natural mRNA produced by the cell to form duplexes. These duplexes then block either the further transcription of the mRNA or its translation.
  • EXAMPLE 1 tumor cells were transfected with a vector comprising full-length human wnt-5& cDNA.
  • the cDNA was subcloned into pRSV (Dr. Jackie Papkoff, Sugen, Redwood City, CA) and orientation determined by restriction analysis.
  • SV-HUC-1 and MC-T16 uroepithelial carcinoma cells were cultured in F12 media supplemented with 1% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin. The media was also supplemented with essential amino acids, ferritin, dexamethasone, insulin, and glutamine as previously described. Cells were grown to 80% confluence before each passage.
  • FBS fetal bovine serum
  • penicillin/streptomycin penicillin/streptomycin
  • passage 20 cells were grown to 50% confluence and the media exchanged for low serum media (Optimem, Gibco).
  • liposomes Lipofectin, Gibco
  • the cells were transfected overnight at 37°C in 5% C0 2 .
  • the media was replaced and the cells grown overnight in media with 1% FBS without the addition of Geneticin (G418, Gibco).
  • the cells were then selected in media supplemented with 800 ug/ml G-4I8. Individual clones were isolated, resistant colonies expanded into cell lines, and maintained in media supplemented with 250 ⁇ g/mi G418 for eventual RNA extraction to determine gene expression of wnt-5a.
  • [ 32 P]dCTP using 1-2 x 10 6 cpm/ml The prehybridization and hybridization solutions consisted of 50% formamide, 4x SSPE, 0.2 mg/ml sheared and boiled salmon sperm DNA, 2.5x Denhardt's, and 1% sodium dodecyl sulfate (SDS). Membranes were washed at room temperature twice in 2x SSC, 1% SDS, followed by several washes in O.lx SSC, 0.1% SDS at 55°C. The membranes were then mounted on 8 x 10 film (Kodak) with an intensifying screen and placed at -80°C up to 5 days.
  • Figure 1A shows the results of the Northern analysis using a random primed wnt-5 ⁇ cDNA 2 P-labeled probe. The results demonstrated expression of the expected 3.2 kb size RNA in several clonally expanded cell lines. Two clones expressing wnt-5 ⁇ shown in Figure 1A were selected for comparison to the MC-T16Ineo transformed and SV-HUC-1/neo nontumorigenic immortal cell lines resistant to G-418.
  • the growth properties were compared between the MC-T16/neo parental cells and two clones of MC-T16/vr ⁇ t-5a to determine whether wnt-5a had any influence on growth kinetics.
  • To determine the growth rates, cell saturation density, population doubling time, and morphologic phenotype of MC-T16/neo cells and MC-T ⁇ 6/wnt-5a cells cells were plated in 12-well dishes at a density of 4 x 10 4 cells per well. The cells were counted every two days for seventeen days, and the growth rate and population doubling time determined from the logarithmic part of the growth curve. The saturation density was determined from the cell number after the cells reached confluence.
  • Morphology was determined by growing cells to confluence and photographed. As shown in Table 1 and Figure IB, the growth rate of the MC-T16/neo cells expressing wnt-5a was similar to MC-T16 cells in the logarithmic phase unlike that for SV-HUC-1/neo cells. That is, the population doubling time of MC-T16/neo cells was 26 hours while that for MC-T16/w «t-5a-clone 6 cells was 32 hours and 24 hours for MC-T16/w «t-5a-clone 900, in comparison to the doubling time for SV-HUC-1 /neo cells which was 56 hours. Cell saturation density at confluence correlated with growth rate.
  • MC-T16/neo parental cells had a saturation density of 8.9 x 10 5 at confluence, while both MC-T16 clones expressing wntSa had saturation densities of 9.4 x 10 5 and 8.0 x 10 5 . This was significantly different than that observed for SV-HUC-1 /neo cells which had a saturation density of 3.2 x 10 5 .
  • Cells at passage 18-20 were seeded at 4 x 10 ⁇ /5 cm plastic dishes for - thymidine incorporation or 12-well plastic dishes for growth kinetics and saturation density. The saturation density was determined on day 13. To determine growth in 0.35% soft agar, 10 ⁇ cells were suspended and colony formation determined at two weeks. The values shown are mean +/- SE of at least three dishes. The experiments were repeated three times with similar results.
  • the parental SV-HUC-1/neo cells characteristically retain many of the features associated with normal epithelial cells in culture.
  • the isolated SV-HUC-1/neo clone used for these experiments no longer grew at confluence unlike that for SV-HUC-1/neo pooled clones which become tightly packed at confluence.
  • MC-T16/neo carcinoma cells continue to grow at confluence, are refractive, and have a spindle cell phenotype. However, when two different clones of MC-T16/w «t-J ⁇ cells were examined, the cells at confluence become less spindle shaped, more flattened, and pleiomorphic, similar to the SV-HUC-1/neo cells.
  • Example 1 the transfected cells of Example 1 were examined in an anchorage dependance assay.
  • 10 4 cells were plated in 0.35% agar (Noble) suspension using standard media over a previously poured 0.5% agar base in 12-well dishes.
  • G-418 resistant MC-T16/neo cells were used as a positive control.
  • Two clones of MC-T 16/w «t-5a were plated for comparison. Colony formation was determined daily for two weeks. Media was added to the wells as needed. Three separate soft agar assays were done as above and the results pooled.
  • MC-TI6-neo cells grow in 0.35% agar with a cloning efficiency of 9.7% compared to no growth in corresponding parental G418 selected SV-HUC-1/neo cells (Table 1).
  • the expression of w «t-5a in MC-T 16 cells re-establishes anchorage dependent growth under these conditions in two different clones. The experiments were repeated three times with similar results.
  • tumorigenesis of wnt-5a transfected tumor cells were examined in vivo.
  • the tumorigenic potentials of G-418 selected positive control MC-T16/neo cells, negative control SV-HUC-1/neo cells and two clones of MC-T16/w «t-5a were tested by inoculations into 4-6 week old female athymic nude mice (Charles River). Mice were housed in sterile bubbles in a temperature and humidity controlled room. Inoculations of 2 x 10 6 cells/site in a total volume of 0.1 ml were made s.c. in the right dorsal quadrant. The animals were examined weekly.
  • Tumors were removed at six months or when necrotic or when 1.5-2 cm in diameter. Representative sections of tumors were fixed in formalin for histologic preparation and stained with H and E. Fixed and stained tumors were examined blindly by three pathologists. Representative pieces of some tumors were used to initiate tumor cell lines using an explant technique. Tumor tissue was cut into 1 mm 2 explant fragments after washing in PBS which were then plated onto tissue culture grade dishes in F-12 1% fetal bovine serum supplemented media without G-418. After I week, the explants were grown in the presence of 250 ⁇ g/ml G-418 and the cells expanded for later analysis.
  • mice inoculated with MC-T16/w «t5a-clone 900 cells grew tumor after a lag time of six weeks. Tumor regression occurred in one animal and one 0.6 cm x 0.8 cm necrotic tumor was removed from another animal. At three months, these tumors grew to a maximum size which never enlarged greater than 1 cm x 1 cm over the following three months (Table 2).
  • mice inoculated with MC-T16/neo cells were attached to Table 2 Tumorigenicity of human uroepithelial cells in athymic nude Cells at passage 18-20 were grown and 2 x 10 ⁇ cells in 0.1 ml were injected s.c into the right dorsal quadrant of 4-6 week old female athymic nude mice.
  • the present invention is not limited to a precise mechanism by which «t-5a is able to suppress tumorigenicity, it is of interest that the MC-T16/w «t-5 ⁇ cells which did form tumors, formed tumors which were relatively avascular with extensive necrosis compared to the parental MC-T16/neo derived tumors. This raises the possibility that wntSa signalling involves pathways important for neo-angiogenesis. Interestingly, the tumors formed from MC-T ⁇ 6/wnt-5a cells also were remarkably devoid of stroma. The relationship between stroma, cell adhesion molecules, and angiogenesis is well documented.
  • telomerase a ribonucleoprotein enzyme complex that adds telomeric repeats (hexanucleotide 5'-TTAGGG-3') to the ends of replicating chromosomes, or telomeres, and that another member of the Wnt family has been shown to regulate telomerase.
  • telomerase activity is compared between two different G-418 resistant clones of parental MC-T16/neo cells and two clones of MC-T16/w «t-5a cells by the primer extension telomere repeat amplification protocol (TRAP) assay in which telomerase synthesizes telomeric repeats onto oligonucleotide primers.
  • TREP primer extension telomere repeat amplification protocol
  • Two different MC-T16/neo cell lines were compared for telomerase activity to Two different MC-T16/w «t-5a cell lines.
  • Subconfluent cultures were used to prepare the detergent 3-[(cholamidopropyl) dimethylaminonio]- 1 -propanesulfonate (CHAPS) extracts.
  • Telomerase enzyme activity was measured by using a PCR-based telomeric repeat amplification protocol (TRAP) kit from Oncor, Inc. as per manufacturer's instructions.
  • Each reaction product was amplified in the presence of an internal TRAP assay standard (ITAS, 36 bp).
  • the TRAP reaction products were separated by 10% PAGE, dried, and autoradiographed.
  • the basal levels of telomerase activity was measured by serial dilution of the protein extracts, and an appropriate range of protein concentration selected that produced a linear response.
  • Each set of TRAP assay included control reaction tubes without any extract, extracts treated with RNase A (200 ⁇ g/ml), and positive assay control TSR8 template (2 ⁇ l/tube).
  • the average optical density of first six TRAP bands after primer band was used as a ratio to ITAS band.
  • the results are shown in Figure 3. The results indicate no change in telomerase activity in either MC-T16/vc «t-5a expressing cell lines.
  • Telomerase activity is believed to correlate with cell immortalization, cell transformation, and with tumorigenesis. Telomerase is a ribonucleoprotein complex which adds telomeric repeats onto chromosomal ends. This is thought to protect the ends against nucleases and ligases thus maintaining stability, and to counter shortening of telomere length as a result of DNA replication. Most human tumor cells have enhanced telomerase activity when compared to normal somatic cells. It has recently been reported that telomerase is activated in wnt- ⁇ mediated mouse mammary tumors. Broccoli et al., "Telomerase activation in mouse mammary tumors: Lack of detectable telomere shortening and evidence for regulation of telomerase RNA with cell proliferation," Mol
  • telomere shortening RNA-binding protein
  • telomerase activity in tumor cells increases during S phase with less activity detected during Go/Gl. It would be of interest to determine whether SV40 alters cell-cycle dependent telomerase activity.
  • human wnt-5 ⁇ is a novel tumor suppressor gene and is likely to be one of the suppressor genes deleted or rearranged on chromosome 3p.

Abstract

L'invention concerne des composés et des méthodes de traitement du cancer, et plus particulièrement, des compositions et des méthodes destinées à transfecter des cellules tumorales dans des conditions telles que la croissance de la tumeur est stoppée. La transfection du gène wnt-5a humain dans des cellules tumorales provoque une diminution de l'action tumorigène, permettant ainsi un traitement anti-cancer ne faisant appel ni à la radiothérapie ni à des agents chimiothérapeutiques toxiques.
PCT/US1997/022087 1996-11-27 1997-11-26 Composes et methodes de traitement du cancer Ceased WO1998023730A1 (fr)

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US6344541B1 (en) 1998-09-25 2002-02-05 Amgen Inc. DKR polypeptides
EP1226172A1 (fr) * 1999-11-05 2002-07-31 Forskarpatent i Syd AB Composes, anticorps et methodes destines au traitement, a la prevention ou au diagnostic de la metastase des tumeurs
US7713526B2 (en) * 2001-05-01 2010-05-11 The Regents Of The University Of California Wnt and frizzled receptors as targets for immunotherapy in head and neck squamous cell carcinomas
WO2002088081A2 (fr) 2001-05-01 2002-11-07 The Regents Of The University Of California Recepteurs wnt et frizzled utilises comme cibles en immunotherapie contre un carcinome spinocellulaire cervico-facial
EP2328604B1 (fr) * 2008-08-13 2014-10-08 Wntresearch AB Utilisation de dérivés peptidiques wnt5-a pour le traitement du mélanome et du cancer gastrique
PL2726880T3 (pl) 2011-07-01 2019-09-30 Wntresearch Ab Leczenie nowotworu prostaty i sposób określania prognozy dla pacjentów z nowotworem prostaty

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