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WO2003006029A1 - Procede visant a induire l'apoptose dans des cellules cancereuses - Google Patents

Procede visant a induire l'apoptose dans des cellules cancereuses Download PDF

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Publication number
WO2003006029A1
WO2003006029A1 PCT/AU2002/000936 AU0200936W WO03006029A1 WO 2003006029 A1 WO2003006029 A1 WO 2003006029A1 AU 0200936 W AU0200936 W AU 0200936W WO 03006029 A1 WO03006029 A1 WO 03006029A1
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igfbp
cell
expression
cells
vector
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PCT/AU2002/000936
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English (en)
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Robert Charles Baxter
Alison Joy Butt
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The University Of Sydney
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Publication of WO2003006029A1 publication Critical patent/WO2003006029A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to a method of inducing apoptosis in cancer cells, a method of killing cancer cells, a method of sensitising cancer cells to agents that induce apoptosis, and a method of treating cancer in a patient.
  • the invention also relates to an adenoviral or other viral vector for increasing the expression of IGFBP-5 by a cell when used in the methods of the invention.
  • IGFs Insulin-like growth factors
  • IGFBPs Insulin-like growth factor binding proteins
  • IGFBP-1 to IGFBP-6 six types of insulin like growth factor binding proteins have been identified. These have been designated IGFBP-1 to IGFBP-6. In addition to binding IGF-I and II, some of these binding proteins exhibit biological effects independent of IGF-I and II.
  • IGFBPs The precise biological actions of IGFBPs are poorly understood. Both stimulatory and inhibiting effects of IGFBPs on cell proliferation have been reported under various experimental conditions.
  • IGFBP-5 Exogenous IGFBP-5 has been reported to have a growth stimulatory effect in normal bone cells (Richman et al . 1999, Endocrinology 140: 4699-4705) . In contrast, Tonner et al . , 2000, Adv. Exp . Med. Biol . 480 : 45-53 have recently shown that IGFBP-5 interacts with IGF- I to prevent IGF-I interaction with the IGF-I receptor in normal mammary epithelial cells in rats and thus leads to cell apoptosis in normal mammary epithelial cells in rats.
  • WO 01/05435 reports that reduction of IGFBP-5 expression in hormone-regulated tumours, for example breast and prostate tumours, using antisense oligodeoxynucleotides targeted to IGFBP-5, results in decreased growth rate of cancer cells.
  • tumour suppressor protein p53 is required for cancer cells to be killed effectively using conventional radiotherapy and chemotherapy.
  • certain tumour cells do not express functional p53 protein and this is a common mechanism by which cancer cells can become refractory to conventional radiotherapy and chemotherapy. This is a significant limitation of conventional therapies. It would be desirable to provide methods of treating cancer using radiotherapy and chemotherapy which are effective in treating tumours which do not express functional p53 protein.
  • IGFBP-5 stimulates growth or inhibits apoptosis in cancer cells
  • the inventors of the present invention have unexpectedly found that apoptosis can be induced in cancer cells by causing the cells to increase the expression of IGFBP-5.
  • the inventors have further found that increasing expression of IGFBP-5 in cancer cells sensitises the cancer cells to apoptosis inducing stimuli, such as radiation and cytotoxic agents.
  • the inventors have surprisingly found that these effects are independent of IGF.
  • the inventors have also surprisingly found that these effects are independent of the p53 phenotype of the cancer cell .
  • the invention provides a method of inducing apoptosis in a cancer cell comprising increasing the expression of IGFBP-5 by the cell to an apoptosis inducing amount.
  • the expression of IGFBP-5 by the cell is increased by introducing into the cell a nucleic acid molecule capable of increasing the expression of IGFBP-5 by the cell.
  • a nucleic acid molecule capable of increasing the expression of IGFBP-5 by the cell is preferably introduced into the cell by contacting the cell with an adenoviral or other viral vector comprising a nucleic acid molecule capable of increasing the expression of IGFBP-5 by the cell.
  • the expression of IGFBP-5 by a cancer cell is increased by contacting the cell with an adenoviral or other viral vector comprising a nucleic acid molecule capable of increasing the expression of IGFBP-5 by the cell, the expression of IGFBP-5 is typically increased to an apoptosis inducing amount .
  • the inventors have also found that increasing the expression of IGFBP-5 by a cancer cell has an effect of ⁇ sensitising' the cell to stimuli that induce apoptosis, such as radiation and cytotoxic agents, so that with the further stimuli, it is possible to induce apoptosis.
  • apoptosis By “sensitising the cell to stimuli that induce apoptosis” , it is meant that a given apoptosis-inducing stimuli (for example, a given dose of radiation) is more likely to induce apoptosis in the cell when compared to a cell not so sensitised.
  • the ability to sensitise a cancer cell to stimuli that induce apoptosis is particularly important for the treatment of tumours which have acquired a level of resistance to apoptosis.
  • the invention provides a method of sensitising a cancer cell to stimuli that induce apoptosis comprising increasing the expression of IGFBP-5 by the cell .
  • the invention provides a method of killing a cancer cell comprising sensitising the cell to stimuli that induce apoptosis by increasing the expression of IGFBP-5 by the cell, and simultaneously or subsequently exposing the cell to apoptosis-inducing stimuli.
  • the invention provides a method of killing a cancer cell comprising exposing the cell to apoptosis-inducing stimuli and simultaneously or subsequently increasing the expression of IGFBP-5 by the cell to an apoptosis inducing amount.
  • the expression of IGFBP-5 by the cell is increased by introducing into the cell a nucleic acid molecule capable of increasing the expression of IGFBP-5 by the cell.
  • a nucleic acid molecule capable of increasing the expression of IGFBP-5 by the cell is preferably introduced into the cell by contacting the cell with an adenoviral or other viral vector comprising a nucleic acid molecule capable of increasing the expression of IGFBP-5 by the cell.
  • the apoptosis-inducing stimuli is radiation or a cytotoxic agent .
  • the methods of the first to fourth aspects of the invention are useful for treating cancer in a patient.
  • the invention provides a method of treating cancer in a patient comprising administering a therapeutically effective amount of a vector for introducing into a cell of the cancer a nucleic acid molecule capable of increasing the expression of IGFBP-5 by the cell to an apoptosis inducing amount.
  • the vector is an adenoviral vector or other viral vector comprising a nucleic acid molecule capable of increasing the expression of IGFBP-5 by the cell.
  • the invention provides a method of treating cancer in a patient comprising administering a therapeutically effective amount of a vector for introducing into a cell of the cancer a nucleic acid molecule capable of increasing the expression of IGFBP-5 by the cell and exposing at least some of the cancer cells to apoptosis-inducing radiation.
  • the vector is an adenoviral vector or other viral vector comprising a nucleic acid molecule capable of increasing the expression of IGFBP-5 by the cell.
  • the vector is administered to the patient prior to exposing the cancer cells to apoptosis-inducing radiation.
  • the cancer cells are exposed to apoptosis-inducing radiation prior to administering the vector to the patient.
  • the invention provides a method of treating cancer in a patient comprising administering a therapeutically effective amount of a vector for introducing into a cell of the cancer a nucleic acid molecule capable of increasing the expression of IGFBP-5 by the cell and exposing at least some of the cells of the cancer to a cytotoxic agent .
  • the vector is an adenoviral vector or other viral vector comprising a nucleic acid molecule capable of increasing the expression of IGFBP-5 by the cell.
  • the vector is administered to the patient prior to exposing the cancer cells to the cytotoxic agent. In other embodiments, the cancer cells are exposed to the cytotoxic agent prior to administering the vector to the patient.
  • a therapeutically effective amount of the vector is an amount of the vector which is sufficient to introduce the nucleic acid molecule into sufficient cancer cells such that the method of treatment inhibits the proliferation of the cancer cells or reduces the number of cancer cells in the patient.
  • the nucleic acid molecule capable of increasing expression of IGFBP-5 by the cell comprises a nucleic acid sequence encoding human IGFBP-5.
  • the patient is a mammal, in particular a human.
  • a significant advantage of the methods of the invention is that the methods can be used to induce apoptosis in cancer cells, kill cancer cells, sensitise cancer cells to apoptosis inducing stimuli, and treat cancer in a patient, independent of the p53 phenotype of the cancer cell.
  • a further advantage of the methods of the invention is that the methods can be used to induce apoptosis in cancer cells, kill cancer cells, sensitise cancer cells to apoptosis inducing stimuli, and treat cancer in a patient, in the absence of IGF.
  • apoptosis may be induced in a cancer cell by increasing expression of IGFBP-5 by the cell in serum free media, or in other words, media which does not contain IGF. This means that the usefulness of the invention is not dependent on IGF expression.
  • the invention provides a method of inducing apoptosis in a cancer cell comprising increasing the expression of IGFBP-5 by the cell to an apoptosis inducing amount in the absence of IGF.
  • the invention provides an adenoviral or other viral vector comprising a nucleic acid capable of increasing the expression of IGFBP-5 by a cell when used in a method of the invention.
  • Figure 1 shows (A) the complete amino acid sequence of human IGFBP-5 and (B) the nucleic acid sequence of human IGFBP-5 cDNA.
  • Figure 2 shows a plot of viable cell numbers over time in the human breast cancer cell line MDA-MB-231 stably transfected with either IGFBP-5 cDNA (triangle) or vector alone (circles) .
  • Cell numbers are shown on the right hand y-axis and the time (days) at which cells were counted is shown on the x-axis.
  • the concentration of IGFBP-5 is indicated on the left hand y-axis and the amount present in conditioned media at each time point is indicated by vertical bars.
  • Figure 3 shows plots of viable cell numbers over time (days) in the breast cancer cell lines MDA-MB-231 (A) and Hs578T (B) infected with IGFBP-5 (triangles) or vector- expressing (circles) adenovirus .
  • Cell numbers are shown on the right hand y-axis and the time (days) at which cells were counted is shown on the x-axis.
  • the concentration of IGFBP-5 is indicated on the left hand y-axis and the amount present in conditioned media at each time point is indicated by a vertical bar.
  • Figure 4 shows a plot of [ 3 H] thymidine incorporation in MDA-MB-231 cells stably transfected with vector (VEC) or IGFBP-5 cDNA (BP-5) or MDA-MB-231 and Hs578T cells post- infection with vector-adenovirus (AdVEC) or IGFBP-5- adenovirus (AdBP-5) .
  • Figure 5 shows a plot of the proportion of IGFBP-5- expressing (black bars) or vector-expressing (white bars) MDA-MB-231 stable transfectants in G0/G1, S or G2/M cell cycle phase 48 h post-seeding.
  • Figure 6 shows a plot of the proportion of IGFBP-5- expressing (black bars) or vector-expressing (white bars) MDA-MB-231 cells in G0/G1, S or G2/M cell cycle phase 48 h post-infection with IGFBP-5 adenovirus or vector- adenovirus .
  • Figure 7 shows a plot of the proportion of IGFBP-5- expressing (black bars) or vector-expressing (white bars) Hs578T cells in G0/G1, S or G2/M cell cycle phase 48 h post-infection with IGFBP-5 adenovirus or vector- adenovirus .
  • Figure 8 shows a plot of the survival of MDA-MB-231 cells stably transfected with vector (white bars) or IGFBP-5
  • Figure 9 shows a plot of the percentage of MDA-MB-231 or Hs578T cells in the pre-Gl peak as determined by flow cytometry 48 post infection with IGFBP-5-adenovirus (black) , vector-adenovirus (white) or IGFBP-5adenovirus + the caspase inhibitor z-VAD-fmk.
  • Figure 10 shows the results of DAPI staining of MDA-MB-231 and Hs578T cells post-infection with vector-adenovirus
  • AdVEC IGFBP-5-adenovirus
  • AdBP-5-adenovirus illustrating nuclear fragmentation. Also shown is a plot of scoring of apoptotic cells identified by DAPI staining.
  • Figure 11 shows a plot of Bax and Bel-2 protein levels in
  • MDA-MB-231 cells stably transfected with vector (white) or IGFBP-5 (black) or MDA-MB-231 and Hs578T cells 48 hr post- infection with IGFBP-5-adenovirus (black) or vector- adenovirus (white) .
  • Figure 12 shows a plot of expression of Bax mRA relative to 36B4 mRNA in MDA-MB-231 cells stably transfected with vector (VEC) or IGFBP-5 cDNA (BP-5) or MDA-MB-231 and Hs578T cells 48 post-infection with vector-adenovirus (AdVEC) or IGFBP-5-adenovirus (AdBP-5) .
  • VEC vector-adenovirus
  • AdBP-5-adenovirus AdBP-5-adenovirus
  • Figure 13 shows [3H] thymidine incorporation to assess the rate of DNA synthesis following 24 hr incubation with or without rhIGF-II (20ng/ml or lOOOng/ml) , or anti-IGFRI antibody IR-3 (lOmg/ml) as indicated in MDA-MB-231 cells stably transfected with vector.
  • Figure 14 shows an immunoblot analysis of IGFBP-5 in 48 hr conditioned media of adenoviral infected MDA-MB-231 and Hs578T cells, probed with antisera to IGFBP-5.
  • Lane 1 full length 30kDa, ; Lane 2, 20 + lOkDa; Lane 3, 20kDa; Lane 4, full length 30kDa; Lane 5, 20 + 10 kDa; Lane 6, adenoviral IGFBP-5 produced by 911 retinoblastoma cells.
  • Figure 15 shows a plot of the proportion of MDA-MB-231 or Hs578T cells in pre-Gl phase following treatment with various forms of IGFBP-5 (black/striped/dotted) purified from conditioned media from the same cell type (MDA-MB-231 or Hs578T cells, respectively), or untreated controls. Hatched bars indicate cells treated with recombinant human IGFBP-5 purified from 911 retinoblastoma cells
  • Figure 16 shows a plot of [ 3 H] thymidine incorporation by MDA-MB-231 or Hs578T cellsfollowing treatment with various forms of IGFBP-5 (black/striped/dotted) purified from conditioned media from the same cell type (MDA-MB-231 or Hs578T cells, respectively), or untreated controls. Hatched bars indicate cells treated with recombinant human IGFBP-5 purified from 911 retinoblastoma cells
  • Figure 17 shows an immunoblot of nuclear (n) and cytoplasmic (c) fractions of MDA-MB-231 cells stably transfected with vector (VEC) or IGFBP-5 cDNA (BP-5) or MDA-MB-231 and Hs578T cells post-infection with vector- adenovirus (VEC) or IGFBP-5-adenovirus (BP-5) probed with 125 I-labelled IGF-II.
  • Figure 18 shows tumour growth rates of tumours derived from MDA-MB-231 cells stably transfected with vector (VEC) or IGFBP-5 cDNA (MDA/BP-5) following injection of the cells into athymic nude mice.
  • Figure 19 shows tumour wet weight of tumours derived from MDA-MB-231 cells stably transfected with vector (MDA/VEC) or IGFBP-5 cDNA (MDA/BP-5) 29 days following injection into athymic nude mice.
  • Figure 20 shows the results of probing northern blots of total RNA extracted from tumours derived from MDA-MB-231 cells stably transfected with vector (MDA/VEC) or IGFBP-5 ' cDNA (MDA/BP-5) with a labelled IGFBP-5 cDNA probe or labelled 36B4 probe (control) .
  • Lane C illustrates total RNA extracted from MDA-MB-231 cells transfected with IGFBP- 5 cDNA as a positive control .
  • Figure 21 shows a plot of the results of real-time quantitative PCR quantification of bax mRNA levels in tumours derived from MDA-MB-231 cells stably transfected with vector (white) or IGFBP-5 cDNA (black) .
  • IGFBP-5" means human or animal IGFBP-5, a fragment thereof that is capable of inducing apoptosis, a polypeptide having an amino acid sequence that is homologous to the IGFBP-5 amino acid sequence of Figure 1A and that is capable of inducing apoptosis, or a fusion between human or animal IGFBP-5 or a fragment thereof and a polypeptide whereby the fusion is capable of inducing apoptosis.
  • a polypeptide is considered to have an amino acid sequence that is homologous to the IGFBP-5 amino acid sequence of Figure 1A, when at least about 70%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% of its amino acid sequence is identical to the IGFBP-5 amino acid sequence of Figure 1 over the defined length of the amino acid sequence.
  • the methods of the invention may be applied to any cancer cell.
  • the cancer cell may, for example, be a breast cancer cell or a prostate cancer cell.
  • the cancer cell may, for example, be an ovarian, lung or colon cancer cell.
  • the expression of IGFBP-5 by the cancer cell is increased relative to the expression of IGFBP-5 by a cancer cell to which the method of the invention has not been applied.
  • the IGFBP-5 for use in the methods of the invention has an amino acid sequence which is homologous to the amino acid sequence of IGFBP-5 shown in Figure 1A and is capable of inducing apoptosis in a cell .
  • the IGFBP-5 for use in the methods of the invention comprises a portion of the amino acid sequence of IGFBP-5 shown in Figure 1A and is capable of inducing apoptosis in a cell .
  • expression of IGFBP-5 by the cancer cell is increased by introducing into the cell a nucleic acid molecule capable of increasing the expression of IGFBP-5 by the cell .
  • the nucleic acid molecule may be introduced by any means known in the art for introducing a nucleic acid molecule into a cell .
  • the nucleic acid molecule may be introduced by, for example, contacting the cell with a plasmid vector or a viral vector (for example, viral vectors derived from adenovirus, retrovirus, poxvirus, herpesvirus, adenovirus-associated virus, etc) comprising the nucleic acid molecule.
  • a plasmid vector for example, viral vectors derived from adenovirus, retrovirus, poxvirus, herpesvirus, adenovirus-associated virus, etc
  • the viral vector is an adenoviral vector which is defective for replication (unable to replicate autonomously in a host cell) .
  • the technology of adenoviruses is described in, for example, Graham and Prevec in Methods in Molecular Biology, 1991, vol 7, pp. 109-128, ed E.J.Murey, The Human Press Inc) .
  • the adenoviral vector is derived from the genome of an adenovirus, and comprises at least the inverted terminal repeats (ITRs) and an encapsidation sequence and lacks all or part of the El adenoviral region.
  • E3 adenoviral region In addition, it can lack all or part of the E3 adenoviral region. However, according to an advantageous embodiment, preference is given to retaining the part of the E3 region which encodes polypeptides, in particular the glycoprotein gpl9k (Gooding et al . , Critical Review of Immunology, 1990, 10:53-71), which make it possible to escape the immune system of the host.
  • the vector may contain additional deletions or mutations which affect, in particular, all or part of one or more regions selected from the E2 , E4 , LI, L2, L3, L4, and L5 regions (see for example WO 94/28152) .
  • the temperature sensitive mutation which affects the DBP (standing for DNA-binding protein) gene of the E2 A region consists in deleting the E4 region with the exception of the sequences which encode open reading frames (ORFs) 6 and 7 (these limited deletions do not require the E4 function to be complemented; Ketner et al . , Nucleic Acids Res., 1989, 17: 3037-3048) .
  • the adenoviral vector is Ad-Track- CMV.
  • the adenoviral vector may be derived from the genome of an adenovirus of human, canine, avian, bovine, murine, ovine, porcine or simian origin or else from a hybrid which comprises adenoviral genome fragments of different origins.
  • the adenoviral vector is of human origin which is preferably derived from a serotype C adenovirus, in particular a type 2 or type 5 adenovirus.
  • the nucleic acid molecule capable of increasing the expression of IGFBP-5 by> the cell comprises a nucleic acid sequence encoding IGFBP-5.
  • the nucleic acid sequence encodes human IGFBP-5 or a fragment thereof that is capable of inducing apoptosis in the cell.
  • the nucleic acid sequence may, for example, be the nucleic acid of Figure IB.
  • the nucleic acid sequence encoding IGFBP-5 may be operably linked to a constitutive promoter, to an inducible promoter or to a promoter that is active only in certain tissues.
  • promoters which may be operably linked to the nucleic acid sequence encoding IGFBP-5 include the CMV (Cytomegalovirus) and RSV (Rous Sarcoma Virus) viral promoters, the promoter of the HSV-1 virus TK gene, the SV40 (Simian Virus 40) virus early promoter, the adenoviral promoter of an early or late (E1A, MLP, etc.) gene, the eukaryotic promoters of the PGK (Phospho Glycerate kinase) , MT (metallothionein) , alpha.1- antitrypsin, CFTR, surfactant (lung-specific) , immunoglobulin (lymphocyte-specific) , actin (muscle- specific) or SRa (hybrid between the SV40 origin and the HTLV-1 LTR; Takebe et al .
  • the promoter can also be a promoter which stimulates expression in a particular type of tumour or cancer cell, or a tumour or cancer cell derived from a particular tissue such as breast tissue, avoiding expression in other cell types. Promoters which may in particular be mentioned are the promoters of the MUC-1 gene, which is overexpressed in breast and prostate cancers (Chen et al . , 1995, J. Clin. Invest. 96, 2775-2782), the CEA (standing for carcinoma embryonic antigen) gene, which is overexpressed in colon cancers (Schrewe et al . , 1990, Mol. Cell. Biol.
  • the promoter is the Cytomegalovirus (CMV) early promoter.
  • CMV Cytomegalovirus
  • the adenoviral vectors for use in the method of the present invention may be propagated in a complementing cell line which is able to supply the defective functio (s) in trans in order to produce the peptides which are required for forming the infectious viral particles.
  • a complementing cell line which is able to supply the defective function (s) in trans in order to produce the peptides which are required for forming the infectious viral particles.
  • cell line 293 for complementing the El function (Graham et al . J. Gen. Virol., 1977, 36: 59-72) or of the cell lines described in WO 97/04119 for effecting a double complementation.
  • the viral particles which are produced are recovered and if need be, purified using the techniques of the art (for example, caesium chloride gradient, chromatographic steps, etc.).
  • the nucleic acid molecule capable of increasing the expression of IGFBP-5 by the cancer cell is inserted into the vector in the place of the deleted adenoviral regions, in particular the El region.
  • the genome of the recombinant adenoviral vector can be prepared by molecular biology techniques or by homologous recombination (see for example WO 96/17070) .
  • the nucleic acid molecule capable of increasing expression of IGFBP-5 by the cell is introduced into the cancer cell by contacting the cancer cell with an infectious adenoviral vector or other viral vector comprising the nucleic acid molecule.
  • the vector may conveniently be contacted with the cancer cells by direct intratumoural injection of the vector.
  • the vector comprising the nucleic acid molecule capable of increasing expression of IGFBP-5 by the cell is integrated into the genome of the cancer cell.
  • the vector comprising the nucleic acid molecule capable of increasing expression of IGFBP-5 by the cell may be autonomously replicating.
  • nucleic acid molecules into a cell may also be used.
  • methods such as transfection including, for example, liposome transfection as described in Goomer et al . , 2000, Clinical Orthopaedics and related research, 379S: S189-S200 or other liposome delivery methods, electroporation, direct DNA injection, or any other means known in the art for introduction of nucleic acid molecules into cells.
  • Apoptosis-inducing radiation is an energy source which causes cell death via apoptosis.
  • Any of the energy sources known in the art which are capable of causing irradiation mediated apoptosis in at least a cell which is not resistant to irradiation mediated apoptosis can be used.
  • the dosages of the energy source for causing irradiation mediated apoptosis are known in the art .
  • the energy source is X-rays.
  • the energy of the X-ray is less than lOGy.
  • cytotoxic agents include for example, paclitaxel, cisplatin, etoposide, paraplatin, bleomycin, plicamycin, doxorubicin, dimethyl triazeno imidazole carboxamide, daunorubicin, cytarabine, procarbazine, 1- ( ⁇ -chloroethyl) -1-nitrosourea (CCNU) , hydroxyurea, melphalan, 1,3-bis ( ⁇ -chloroethyl) - 1- nitrosourea (BCNU) , vincristine, vinblastine, o,p' - dichloro-diphenyldicloroethane (o,p'-DDD) (mitotane) , cyclophosphamide, ifosfamide, 5-fluorouracil, buslfan,
  • cytotoxic agents include interferon, tamoxifen, testolactone, L- asparaginase, progesterone, androgens, estrogens and the like. These cytotoxic agents can be administered in about the dosage and amount known in the art .
  • the vector for introducing into a cell of the cancer a nucleic acid molecule capable of increasing the expression of IGFBP-5 by the cell is administered to the patient in a composition comprising the vector and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier is a pharmaceutically acceptable solvent, suspending agent or vehicle for delivering the vector to the patient.
  • the carrier may be liquid or solid and is selected with the planned manner of administration in mind.
  • Suitable pharmaceutically acceptable carriers include fluid and nutrient replenishers.
  • Other suitable pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in Remington's Pharmaceutical Sciences, 15th ed. Easton: Mack Publishing Co., 1405-1412,1461-1487 (1975) and The National Formulary XIV. , 14th ed. Washington: American Pharmaceutical Association (1975) .
  • the pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art. See Goodman and Gilman's The Pharmacological Basis for Therapeutics (7th ed.) .
  • composition may be adapted for oral or parenteral administration.
  • parenteral as used herein includes subcutaneous injections, aerosol, intra-tumoural, intravenous, intramuscular, intrathecal, intracranial, injection or infusion techniques, intra-arterial, intraperitoneal , subcutaneous, intracavity, or transdermal .
  • compositions for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • Suitable non-aqueous carriers for such compositions include non-aqueous solvents such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Suitable aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Other suitable carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, anti-microbials, anti-oxidants, chelating agents, growth factors and inert gases and the like.
  • compositions for oral administration may, for example, be in the form of hard gelatin capsules wherein the vector is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin. They may also be in the form of soft gelatin capsules wherein the vector is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin.
  • water or an oil medium such as peanut oil, liquid paraffin or olive oil.
  • compositions are preferably prepared and administered in dose units.
  • Solid dose units include tablets, capsules and suppositories.
  • different daily doses can be used for treatment of a patient, depending on the manner of administration, nature and severity of the disorder, age and body weight of the subject.
  • the administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals.
  • animal models may be used to determine effective dosages for treatment.
  • Various considerations are described, eg., in Langer, Science, 249: 1527, (1990). It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific composition and vector employed, the age, body weight, general health and sex of the patient, the patients diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy, and will be ultimately determined by the attending physician.
  • compositions may additionally include other pharmaceutically active compounds, such as cytotoxic agents, to provide an operative combination.
  • other pharmaceutically active compounds such as cytotoxic agents
  • the human breast cancer cell lines MDA-MB-231 and Hs578T were routinely maintained in RPMI-1640 supplemented with 10% FCS, 10 ⁇ g/ml insulin and 2.92 mg/ml glutamine under standard conditions.
  • MDA-MB-231 cells were stably transfected with a 0.9kb IGFBP-5 cDNA fragment in the expression vector pOP13 (Invitrogen Corp., Carlsbad, CA) as described in Firth et al ,1998, Biochem. Biophys . Res. Commun. 246:325-329.
  • the cDNA fragment encoded human IGFBP-5 having the amino acid sequence as shown in Figure 1A.
  • Cells were stably transfected using Lipofectamine (Life Technologies, Gaithersberg, MD) according to the manufacturer's protocol IGFBP-5-transfectants (MDA/BP-5) and vector (MDA/VEC) controls were selected in media containing 800 ⁇ g/ml of geneticin for 21 days post-transfection. A mixed population of transfectants was grown up for subsequent experiments .
  • IGFBP-5 expressing cells and control vector expressing cells were seeded out at 5xl0 4 cells per well in a 12 well plate in 10% fetal calf serum media. At 3 , 6, 9 and 13 days post-seeding cells were trypsinised and viable cell number (as assessed by trypan blue exclusion) determined by cell counting using a haemocytometer . Results of the means of triplicate wells from two independent experiments are shown in Figure 2. Conditioned media was also collected at each time point and analysed for levels of IGFBP-5 by specific radioimmunoassay (RIA) . Stable expression of IGFBP-5 resulted in significantly decreased growth rate of MDA-MB-231 cells compared to vector controls (p,0.03) ( Figure 2) .
  • RIA radioimmunoassay
  • the experiments referred to in B above were extended using an adenoviral -mediated expression system to transiently infect human breast cancer cells with IGFBP-5.
  • the human IGFBP-5 cDNA was sub-cloned into the adenoviral shuttle vector, pAd-Track-CMV and replication deficient IGFBP-5 expressing adenoviruses were produced essentially as described in Raffo et al ,2000, Oncogene 19 : 6216-6228; Firth et al . ,1999, Protein Exp. Purif. 16: 202-211.
  • infected cells express green fluorescent protein (GFP) , which can be used as a marker of infection proficiency by determining the percentage of GFP-positive cells using flow cytometry.
  • GFP green fluorescent protein
  • Proliferating cultures of MDA- MB-231 and Hs578T cells were incubated with varying concentrations of adenovirus stock (approximately 4.5 x 10 12 plaque forming units/ ⁇ l) for 6 hours in serum-free, insulin-free media (SF media) . Virus was then removed and replaced with fresh growth media. For cell proliferation assays, cells were infected with 0.1 ⁇ l of adenovirus stock.
  • Hs578T cells were infected with 2 ⁇ l of adenovirus stock, and MDA-MB-231 cells were infected with 1 ⁇ l of adenovirus stock. These concentrations were shown to give >90% infection efficiency and low toxicity as assessed by flow cytometry (data not shown) .
  • IGFBP-5 secreted by vector expressing cells remained at approximately 9 ng/ml (data not shown) .
  • Levels of IGFBP-5 secreted by IGFBP-5-adenovirus infected cells remained significantly elevated over the growth period (left-hand axis) ( Figure 3A and 3B) .
  • Expression of IGFBP-5 by both MDA-MB-231 and Hs578T cells resulted in a significant inhibition of growth compared to cells infected with vector alone adenovirus (p ⁇ 0.0005, p ⁇ 0.005 respectively) .
  • MDA-MB-231 and Hs578T cells were plated in 48 well plates at 5 x 10 /well and infected with adenovirus as described above. After 20 hours incubation in SF media, l ⁇ Ci/well [ 3 H] thymidine (35 Ci/mmol, ICN) was added in 50 ⁇ l of medium for a further 4 hour incubation at 37 °C. Monolayers were rinsed twice with ice-cold saline and fixed with 1 ml/well ice-cold methanol : acetic acid (3:1) at 4°C for a minimum of 2 hours.
  • the percentage of cells with a hypodiploid DNA content was determined using a Coulter ELITE flow cytometer (Coulter, Hialeah, FL) .
  • Figures 5, 6 and 7 show the mean results from at least three independent experiments .
  • Flow cytometry was also used to determine the number of MDA-MB-231 and Hs578T cells with a greater than G2/M DNA content when cells were infected with IGFBP-5 or vector control adenovirus ( Figure 5, 6 and 7) . Twenty thousand cells were analysed for each sample.
  • Both floating and adherent cells were analyzed for induction of apoptosis by flow cytometry. 24 h after plating or post-adenoviral infection, cells were rinsed with fresh SF media then incubated for 48 h with or without the caspase-inhibitor z-VAD-fmk (100 ⁇ M; Bachem AG, Switzerland) . Floating and attached cell populations were combined and 1 x 10 6 cells were prepared and analyzed as described above. Labelled nuclei were gated on light scatter to remove debris and the percentage of nuclei with a sub-Gl content was determined (see Figure 9) .
  • Apoptosis assay Cells were stained with the DNA stain DAPI and nucleic acid fragmentation visualised by fluorescent microscopy. Cells were plated at 5 x 10 5 per well in 6-well plates for 24 h. Cells were then washed with fresh SF media and incubated for 48 h. Attached and floating cells were then pooled, rinsed with PBS, fixed in ice-cold methanol for 10 min and then stained with 0.8 ⁇ g/ml 4, 6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis, MO) . The percentage of apoptotic cells was determined microscopically as cells with visible nuclear fragmentation. Cells infected with vector showed intact nuclei while those infected with IGFBP-5 adenovirus exhibited fragmented nuclei (see Figure 10) .
  • Giant cells which are associated with induction of apoptosis and are reported to occur by disruption of cell cycle checkpoints normally regulated by p2 ⁇ WAFI/CIP1 leading to endoreplication, were observed in DAPI stained cells infected with IGFBP-5 adenovirus but not with cells infected with vector adenovirus.
  • Protein samples were prepared from 1 x 10 6 cells 48 h post- seeding for stable transfectants or post-infection with adenovirus following an incubation in SF media. Samples were resolved under reducing conditions on 12% SDS- polyacrylamide gels using standard methods. Resolved proteins were transferred to nitrocellulose membranes and probed with either anti-Bax polyclonal (1:1000 dilution; PharMingen, San Diego, CA) or anti-Bcl-2 monoclonal (0.8 ⁇ g/ml; Dako, Carpinteria, CA) antibodies overnight at 4°C.
  • the ribosomal protein 36B4 was used as a loading control. The results are shown in Figure 12 and the bars represent the means of bax mRNA levels from two experiments quantitated densitometrically in relation to the control gene 36B4 and expressed as a percentage of the corresponding vector levels .
  • MDA-MB-231 cells stably transfected with vector (MDA/VEC) to the mitogenic effects of IGFs were determined by assessing the rate of DNA synthesis as determined by [ 3 H] thymidine incorporation following 24 h incubation with or without rhIGF-II (20ng/ml or lOOng/ml) , or anti-IGFRI antibody ⁇ IR-3 (lO ⁇ g/ml) .
  • Figure 13 shows the means of quadruple wells from two independent experiments .
  • MDA-MB-231 and Hs578T cells were infected with adenovirus as described above then incubated in SF media for 48 h.
  • Conditioned media was collected and IGFBP-5 was purified using an IGF-I affinity column followed by high performance liquid chromatography.
  • IGFBP-5 immunoblots membranes were incubated with chicken anti-human IGFBP-5 antibody followed by anti-chicken IgY secondary antibody conjugated with alkaline phosphatase and visualised with colorimetric detection (see Figure 14) .
  • the effects of IGFBP-5 obtained from conditioned media on cell cycle and [ 3 H] thymidine uptake were assessed as described above and the results are shown in Figures 15 and 16.
  • MDA-MB-231 cells stably transfected with vector (MDA/VEC) or IGFBP-5 cDNA (MDA/BP-5) were trypsinized and resuspended at 1 x 10 7 viable cells/100 ⁇ l in serum-free media.
  • Cell suspensions were mixed with an equal volume of Matrigel (BD Biosciences, Bedford, MA) as a support matrix for initial cell growth.
  • 1 x 10 7 cells were injected subcutaneously at the dorsal neck into two groups (MDA/VEC and MDA/BP-5) of six mice (in the repeat experiment, seven mice were injected with MDA/BP-5 cells) .
  • tumour growth was monitored weekly by measuring tumour length and width using calipers, and tumour volume was calculated using the formula: length x width 2 x 0.5. Experiments were terminated when tumours reached approximately 1cm 3 . Blood samples were taken from all animals at termination and tumors were frozen in liquid nitrogen for RNA analysis. Results were pooled from two separate experiments (see Figure 18) . Mean wet weight of tumours from all animals with detectable tumours is shown in Figure 19.
  • An IGFBP-5 cDNA probe was labeled using a HexaLabel DNA labeling kit (MBI Fermentas, Vilnius, Lithuania) and [ ⁇ - 32 P] deoxy-CTP . Filters were hybridized at 42°C overnight, then washed in 1 x SSC (standard saline citrate) at 42°C, followed by a wash in 0.1 x SSC heated to 42°C.
  • the ribosomal phosphoprotein, 36B4 was used as a loading control -(Masiakowsk-i -et -a-1—.-,- 1982-, Nucl . Acids Res. 10: 7895-7903). Filters were quantitated using a Phosphorlmager FLA3000 (Fuji, Japan). Data were normalized by expressing the ratio of IGFBP-5 mRNA to 36B4 mRNA (see Figure 20) .
  • RNA Real time quantitative PCR Total RNA was isolated from tumours derived from MDA-MB-231 cells stably transfected with vector or MDA-MB-231 cells and reverse transcribed using oligo dT17 and Superscript II RT-polymerase as previously described (Butt et al . , 2000, J. Biol. Chem. 275: 39174-39181).
  • A. Stable and adenoviral-mediated expression of IGFBP-5 is growth inhibitory to cancer cells.
  • IGFBP-5 The effects of IGFBP-5 on the growth of cancer cells was examined using both stable transfection of human IGFBP-5 cDNA ( Figure 2) as well as transient adenoviral-mediated expression ( Figure 3) .
  • Cell growth was assessed by cell counting over a 13 -day period, and concentrations of IGFBP- 5 secreted into the media were also determined at each time point. Secretion of IGFBP-5 remained high throughout the 13 -day growth period and was low or undetectable in vector control cells (data not shown) .
  • Figure 2 shows the effects of stable expression of IGFBP-5 on the growth of MDA-MB-231 cells.
  • MDA-MB-231 cells stably transfected with IGFBP-5 cDNA were significantly growth inhibited over the 13 -day growth- -period -compared- -to vector ⁇ con-t-ro-1-s (-p-- ⁇ - 0,0-3—-by repeated measures ANOVA) .
  • Similar results were observed with adenoviral-mediated expression of IGFBP-5 in MDA-MB- 231 cells ( Figure 3A) and Hs578T cells ( Figure 3B) .
  • transient overexpression of IGFBP-5 significantly inhibited cell growth compared to vector controls (p ⁇ 0.0005 and p ⁇ 0.005 respectively by repeated measures ANOVA) .
  • IGFBP-5 expression induces a G2/M cell cycle arrest in cancer cells.
  • FIG. 5 demonstrates that stable expression of IGFBP-5 in MBA-MB-231 cells results in a small but significant accumulation in G2/M phase of the cell cycle compared to vector controls (p ⁇ 0.05) .
  • Adenoviral-mediated expression of IGFBP-5 also resulted in a significant increase in the percentage of MDA-MB-231
  • IGFBP-5 expression induces caspase-dependent apoptosis via modulation of Bcl-2 proteins.
  • the effect of stable IGFBP-5 expression on the long-term survival of MDA-MB-231 transfectants was examined following exposure to IR. Cells were irradiated with various doses of x-rays, then radiosensitivity was- ev-a-1-uated- using- clonogenic survival assays. At doses of 2.5 and 5 Gy x- rays, MDA/BP-5 cells showed significantly reduced long-term (up to 14 days) survival (p ⁇ 0.0001 and p ⁇ 0.002 respectively) compared to vector controls ( Figure 8) .
  • the surviving fraction was low in both MDA/VEC and MDA/BP-5 cells.
  • the effect of IGFBP-5 on the induction of apoptosis was examined in MDA-MB-231 and Hs578T cells using transient adenoviral-mediated expression. The extent of DNA fragmentation characteristic of apoptotic cell death was assessed using both flow cytometric analysis of the hypodiploid (pre-Gl) fraction and scoring of DAPI-stained nuclei for fragmentation.
  • Figure 9 illustrates that expression of IGFBP-5 resulted in a significant increase in the percentage of the population in the pre-Gl fraction compared to vector controls in both MDA-MB-231 (from 2.7% + 0.5 to 7.3% ⁇ 1.1, p ⁇ 0.02) and Hs578T (from 2.9% ⁇ 0.8 to 9% ⁇ 3.3, p ⁇ 0.006) cells.
  • Incubation of IGFBP-5- expressing MDA-MB-231 and Hs578T cells with the caspase inhibitor z-VAD-fmk resulted in a significant decrease in the level of apoptosis (p ⁇ 0.01 and p ⁇ 0.02 respectively) to levels which were not significantly different from vector controls ( Figure 9) .
  • IGFBP-5 in both MDA-MB-231 and Hs578T cells is associated
  • IGFBP-5 in MDA-MB-231 cells resulted in a significant downregulation of Bcl-2 protein compared to vector controls
  • Figure 12 shows the levels of bax mRNA in IGFBP-5- expressing MDA-MB-231 and Hs578T cells and vector controls. Densitometric analysis demonstrated that there was a significant increase in bax mRNA levels in IGFBP-5- expressing cells compared to vector controls ( Figure 12; p ⁇ 0.03 stable MDA, p ⁇ 0.04 adenoviral MDA and Hs578T) . Also shown is the level of IGFBP-5 mRNA in MDA-MB-231 and Hs578T cells showing that both stable transfection with IGFBP-5 cDNA and infection with IGFBP-5-adenovirus, resulted in an upregulation of IGFBP-5 mRNA compared to controls ( Figure 11) .
  • the inventors examined whether the growth inhibitory and proapoptotic effects of IGFBP-5 in cancer cells were mediated via IGFBP-5 's ability to sequester IGFs and ablate their mitogenic and anti-apoptotic effects.
  • the response of MDA-MB-231 cells stably transfected with vector (MDA/VEC) to the mitogenic effects of IGFs was determined by examining levels of [ 3 H] thymidine incorporation in the presence or absence of up to 100 ng/ l rhIGF-II.
  • Figure 13 illustrates that MDA/VEC cells were unresponsive to IGF-II.
  • IGFs act through the IGFRI (Baserga et al, 1997, Biochem. Biophys . Acta.
  • IGF-responsive T47D human breast cancer cells 10 ⁇ g/ml ⁇ IR-3 can reverse the mitogenic effects of 20 ng/ml IGF-II (Butt et al . , 2002, Endocrinology 143: 2693-2699).
  • Hs578T cells have been previously demonstrated to be unresponsive to the mitogenic and anti-apoptotic effects of IGFs (Oh et al., 1993, J. Biol. Chem. 268: 14964-14971). These data suggest that the inhibitory and proapoptotic effects of IGFBP-5 cancer cells are unlikely to be mediated via sequestration of IGFs and are independent of IGFRI signalling.
  • IGFBP-5 The proapoptotic effects of IGFBP-5 are not mediated by secreted IGFBP-5.
  • MDA-MB-231 or Hs578T cells were infected with IGFBP-5- adenovirus and incubated in SF media for 48 h. Conditioned media was collected, purified through an IGF-affinity column and fractionated by reverse-phase HPLC. Fractions were separated by electrophoresis, and immunoblotted with an anti-IGFBP-5 antibody.
  • Figure 14 shows that media conditioned by MDA-MB-231 cells infected with IGFBP-5 adenovirus (MDA/AdBP-5) contained some full-length (30 kDa) IGFBP-5, but the majority of immuno-reactive IGFBP-5 was proteolysed into fragments of 20 kDa and 15 kDa.
  • Conditioned media from Hs578T cells infected with IGFBP-5 Hs/AdBP-5) contained predominantly full-length IGFBP-5 but proteolysed forms were also detected ( Figure 14) .
  • IGFBP-5 is inhibitory to the growth of cancer cells in vivo.
  • MDA-MB-231 cells stably transfected with IGFBP-5 (MDA/BP-5) or vector (MDA/VEC) were injected into groups of twelve or thirteen female nu/nu mice respectively, and tumour size was measured weekly up to 4 weeks post-injection when some tumours had reached approximately 1 cm 3 .
  • Tumours derived from MDA/BP-5 had a significantly decreased growth rate (as judged by tumour volume) compared to MDA/VEC tumours ( Figure 18; p ⁇ 0.0001) .
  • RNA from MDA/BP- 5 cells as a positive control for IGFBP-5 cDNA expression ( Figure 21, lane C) was also examined. Serum samples were analyzed for IGFBP-5 expression using an IGFBP-5-specific RIA but levels were too low to be detected by this method.
  • IGFBP-5 Inhibitory effects of IGFBP-5 on the growth of MDA-MB- 231 cells in vivo are associated with an induction of bax mRNA and decrease in bcl-2 mRNA.
  • IGFBP-5-induced growth inhibition in vivo was investigated, by examining the mRNA levels of the proapoptotic regulators, bax and bcl-2 in MDA/BP-5- and MDA/VEC-derived tumours using real-time PCR analysis (Figure 21) .
  • Levels of mRNA were expressed as a ratio of the control gene actin and as a percentage of the levels in MDA/VEC-derived tumours.
  • IGFBP-5 To address the role of IGFBP-5 in the growth of cancer cells, the inventors have examined and characterized the effects of IGFBP-5 on the growth of human breast cancer cells both in vi tro and in vivo .
  • the human breast cancer cell lines MDA-MB-231 and Hs578T both secrete low levels of IGFBP-5 ( ⁇ 5 ng/ml) .
  • Stable transfection of MDA-MB-231 cells with IGFBP-5 cDNA resulted in an increase in secreted levels to approximately 40 ng/ml.
  • Other cancer cell lines secrete similar or higher levels than this (eg. MCF-7 cells secrete ⁇ 30 ng/ml, T47D cells secrete ⁇ 200 ng/ml) , emphasizing the relevance of this model in studying the effects of IGFBP-5 on cancer cell growth.
  • IGFBP-5 expression reduced the survival of human breast cancer cells following the apoptotic stimulus of IR, and resulted in the induction of a caspase-dependent pathway of apoptosis. Furthermore, expression of IGFBP-5 both in vi tro and in vivo induced a transcriptional upregulation of the proapoptotic regulator bax, suggesting IGFBP-5 may induce a mitochondrial apoptotic pathway. These results demonstrate that IGFBP-5 has intrinsic antiproliferative and proapoptotic effects in cancer cells and modulates expression of Bcl-2-like proteins, possibly by direct transcriptional upregulation of proapoptotic genes. These results are in contrast to studies by Perks et al .
  • IGFBP-5 expression appears to be independent of IGF signaling, as neither MDA-MB-231 or Hs578T cells (Oh et al., 1993, J. Biol. Chem. 268: 14964-14971) are responsive to the mitogenic effects of IGFs. Furthermore, the observation that exogenous IGFBP-5 secreted by IGFBP-5- transfectants has no growth effects would suggest it is not acting through a cell-surface receptor in these cells.
  • IGFBP-5 expression was observed in the nuclear compartment of MDA-MB-231 and Hs578T cells and expression resulted in a transcriptional upregulation of bax mRNA.
  • Both MDA-MB-231 and Hs578T cells express mutant, nonfunctional p53 protein (Runnebaum et al . , 1991, Proc . Natl . Acad. Sci. USA 88: 10657-10661). The latter plays a critical role in effecting the cellular response to DNA damage, mediating both a cell cycle arrest and induction of apoptotic cell death (Lowe et al . , 1993, Nature 362: 847- 849) .
  • the inventors' studies demonstrate that IGFBP-5 can induce p53 -independent apoptotic and growth-inhibitory pathways " in cancer cells . This has particular relevance- to the treatment of breast cancers, which frequently harbour inactivating mutations in p53 (Osborne et al . , 1991, Cancer Res. 51: 6194-6198) leading to increased resistance to radio- and chemo-therapy.

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Abstract

L'invention concerne un procédé visant à induire l'apoptose dans une cellule cancéreuse, qui comporte l'étape consistant à accroître l'expression d'IGFBP-5 par la cellule à une quantité induisant l'apoptose ; un procédé visant à tuer des cellules cancéreuses, un procédé de sensibilisation de cellules cancéreuses à des agents induisant l'apoptose, et une méthode de traitement du cancer chez un patient.
PCT/AU2002/000936 2001-07-13 2002-07-15 Procede visant a induire l'apoptose dans des cellules cancereuses WO2003006029A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7192738B2 (en) 2003-10-03 2007-03-20 Genentech, Inc. IGF binding proteins
US7846730B2 (en) * 2001-07-17 2010-12-07 Isis Pharmaceuticals, Inc. Antisense modulation of BCL2-associated X protein expression
EP2799546A4 (fr) * 2011-12-30 2015-07-01 Korea Inst Radl & Med Sciences Marqueur diagnostique d'exposition à un rayonnement igfbp-5, composition permettant de diagnostiquer l'exposition à un rayonnement par mesure du niveau d'expression du marqueur, kit de diagnostic d'exposition à un rayonnement comprenant la composition, et méthode permettant de diagnostiquer l'exposition à un rayonnement faisant appel audit marqueur
EP3082840A4 (fr) * 2013-12-20 2017-11-22 The General Hospital Corporation Méthodes et dosages biologiques se rapportant à des cellules tumorales circulantes

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992003471A1 (fr) * 1990-08-28 1992-03-05 Chiron Corporation Nouvelle proteine de liaison de facteur de croissance analogue a l'insuline igfbp-5
WO2001005435A2 (fr) * 1999-07-19 2001-01-25 The University Of British Columbia Therapie antisens pour tumeurs a regulation hormonale

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992003471A1 (fr) * 1990-08-28 1992-03-05 Chiron Corporation Nouvelle proteine de liaison de facteur de croissance analogue a l'insuline igfbp-5
WO2001005435A2 (fr) * 1999-07-19 2001-01-25 The University Of British Columbia Therapie antisens pour tumeurs a regulation hormonale

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7846730B2 (en) * 2001-07-17 2010-12-07 Isis Pharmaceuticals, Inc. Antisense modulation of BCL2-associated X protein expression
US7192738B2 (en) 2003-10-03 2007-03-20 Genentech, Inc. IGF binding proteins
US7348154B2 (en) 2003-10-03 2008-03-25 Genentech, Inc. IGF binding proteins
US7351545B2 (en) 2003-10-03 2008-04-01 Genentech, Inc. IGF binding proteins
EP2799546A4 (fr) * 2011-12-30 2015-07-01 Korea Inst Radl & Med Sciences Marqueur diagnostique d'exposition à un rayonnement igfbp-5, composition permettant de diagnostiquer l'exposition à un rayonnement par mesure du niveau d'expression du marqueur, kit de diagnostic d'exposition à un rayonnement comprenant la composition, et méthode permettant de diagnostiquer l'exposition à un rayonnement faisant appel audit marqueur
EP3082840A4 (fr) * 2013-12-20 2017-11-22 The General Hospital Corporation Méthodes et dosages biologiques se rapportant à des cellules tumorales circulantes
AU2014364520B2 (en) * 2013-12-20 2020-01-02 The General Hospital Corporation Methods and assays relating to circulating tumor cells
US10900083B2 (en) 2013-12-20 2021-01-26 The General Hospital Corporation Methods and assays relating to circulating tumor cells
AU2020200114B2 (en) * 2013-12-20 2022-01-06 The General Hospital Corporation Methods and assays relating to circulating tumor cells

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