MXPA98001704A - Protein 3 linkage to the insulin-like growth factor (igf-bp3) in the treatment of tumors related to - Google Patents

Abstract

The present invention relates to methods for treating tumors related to p53 by administering (1) an IGF-BP3 modulator, wherein the modulator up-regulates the expression or activity of IGF-BP3, (2) IGF-BP3 itself, or ( 3) an expression vector comprising a nucleotide sequence encoding IGF-BP3. In this latter method, the nucleotide sequence of IGF-BP3 can also be operably linked to an inducible promoter or enhancer, wherein the method further comprises administering an inducer capable of up-regulating or initiating expression of the protein. Additionally, any of the above methods may include as an additional step, the administration of a cytotoxic agent. These methods are specific examples of a broader method. The treatment of tumors related to p53 by inhibiting the binding of IGF to IG

Description

LINKING PROTEIN 3 TO THE INSULIN SIMILAR GROWTH FACTOR (IGF-BP3) IN THE TREATMENT OF TUMORS RELATED TO P53 The present invention relates to methods for using a protein designated IGF-BP3, which affects apoptosis and tumor suppression. This invention also relates to methods for identifying and using IGF-BP3 modulators and mimetics. P53 is a tumor suppressor well known in the art. Up-regulation and activation of p53 are an important cellular response to the genotoxic resistance and deregulated expression of certain oncogenes. Hartwell et al. (1994), Science 266: 1821-8. Accordingly, p53 is required for cell cycle arrest at the Gl checkpoint. Har ell et al. (1994); Kuerbitz et al. 1992), Proc. Nati Acad. Sci USA 89: 7491-5; Kastan et al. (1991), Cancer Research 51: 6304-11. Alternatively, p53 in many cases is required as a binding to apoptosis in response to certain anti-cancer agents and? -irradiation. Yonish-Rouach et al. (1991), Matare 352: 345-7; Shaw et al. (1992), Proc. Nati Acad. Sci USA 89: REF .: 26925 4495-9; Lo e collaborators (1993), Nature 362: 849-52; Lowe and collaborators (1994), Cell 74: 957-67. P53 could also be required as a binding to apoptosis in response to the expression of the oncoprotein E1A and c-myc. Debbas y colaborades (1993), Genes _ & Development 7: 546-54; Lowe et al. (1993), Genes & Development 7: 535-45; Evan et al (1992), Cell 69: 119-28; Hermeking & Eick (1994), Science 265: 2091-3. The tumor suppressor function of p53 is thought to be mediated, at least in part, by its ability to act as a specific transcriptional activator of the sequence. Genes such as p21 / AFl (El-Deiry et al. (1993), Cell 75: 817-25) and GADD45 (Kastan et al. (1992), Cell 71: 587-97) are objective genes regulated by p53. These genes encode proteins that interact directly with the cell cycle components and the DNA replication machinery and provide a direct link between the p53-dependent Gl checkpoint in the cell cycle, DNA repair and cell proliferation. Harper et al (1993), cell 75: 805-16; Xiong, et al. (1993), Nature 366: 701-4;; Smith et al. (1994), Science 266: 1376-80.

Insulin-like growth factors (IGF-1 and -II), together with their receptors (IGFR), promote the growth of tumor cells. IGF-I (and to some extent IGF-II) is a mitogen that stimulates cell proliferation (associated with elevated cyclin DI and cdc2.) And transformation. More recently, studies suggest that IGF-I acts as a survival factor, protecting cells that suffer from apoptosis (cell death). Sell, C. et al. (1995), Cancer Research 55: 303-6. This latter activity may be particularly important in promoting the growth of tumor cells. Animal studies clearly suggest a role for IGF-I and IGF-IR in tumor growth. Protein 3 binding to insulin-like growth factor (IGF-BP3) regulates the axis of IGF-IGFR. Cubbage et al. Recently described 8.9 kb of the genomic sequence, including the promoter and 1.9 kb of the 5 'flank sequence, five exons, four introns and approximately 1.5 kb of the 3 r flank sequence for the IGF-BP3 gene. Cubbage et al. (1990), J. Biol. Chem. 265: 12642-9. This publication is incorporated by reference, including the sequence of IGF-BP3. The technique describes the use of IGF-BP3 in combination with IGF for the treatment of catabolic conditions (eg, burns, trauma, peptic ulcers). International Patent Application WO 9404030. The technique does not disclose, however, a link between the IGF-binding activity of IGF-BP3 and the p53 tumor suppressor. The present invention relates to methods for treating tumors related to p53. The term "related to p53" refers to tumor cells in which wild-type p53 (wt) is absent, disabled or mutated in another way. These treatment methods comprise administering an effective amount of either (1) an IGF-BP3 modulator, (2) or the IGF-BP3 itself, or (3) an expression vector comprising a nucleotide sequence encoding for IGF-BP3. In method (1), the modulator upregulates the expression or activity of IGF-BP3. In method (3), the nucleotide sequence of IGF-BP3 can also be operably linked to an inducible promoter or enhancer, and the method further comprises administering an inducer capable of up-regulating or initiating expression of the protein. Additionally, any of the above methods may include as an additional step the administration of a cytotoxic agent, since the present inventors believe that IGF-BP3 can make tumor cells more susceptible to these agents. The above methods inhibit the binding of IGFs to IGFRs. In this way, these methods are specific examples of a broader method: the treatment of tumor related to p53 by inhibiting the binding of IGF to IGFR. The invention further relates to methods for identifying substances useful in the treatment of tumors related to p53. One such method uses a reporter gene operably linked to the p53-responsive elements described below, wherein the expression of the indicator signals the upregulation of IGF-BP3. Another such method employs a cancer cell that has IGF-I or II receptors in the presence of bases or nucleotides having a detectable label, wherein a decrease in the admission of the label signals a decrease in DNA synthesis directed by the IGF.

Figure la-d shows the regulation of IGF-BP3 gene expression by wild-type p53. The Figure shows the genomic structure of the IGF-BP3 gene indicating the map location (nucleotides 8095-8452) and the nucleotide sequence (SEQ ID No: 1) of the cDNA fragment, V9 probe. Figure Ib shows a comparative analysis by Northern blotting of the expression of p53-induced IGF-BP3 mRNA in EB-1 cells activated with CdCl2. In Figure 1, a temperature-sensitive mutant of p53 (p53V143A) induces the expression of IGF-BP3 mRNA in clonal Saos-2-D4H cells described in Buckbinder et al. (1994), Proc. Nati Acad. Sci. USA 91: 10640-4. Figure 1 shows the kinetic analysis of the expression of the transcript regulated by p53 in EB-1 cells activated with CdCl2. Expression was inspected by Northern blot analysis, normalized to actin expression, and expression quantified by phosphoformation image analysis (Fuji image phosphoformer). Figure 2 shows the characterization of the p53-sensitive DNA elements and of p53-binding in the IGF-BP3 gene. Figure 2a shows two sequences (SEQ ID NOS .: 2 and 3) in the structure of the published IGF-BP3 gene that was determined by computer analysis to have similarity to the p53 consensus binding site (RRRCWWGYYY) 2 (SEC ID NO: 4). These sequences denote here Sequence A (SEQ ID NO: 2) 4 and Sequence B (SEQ ID NO: 3) that appear in the first and second introns, respectively. Figure 2b shows the specific binding of p53 to DNA to Sequence A and B. In Figure 2c, the DNAs of sequence A and B confer inductibility to p53 to a heterologous promoter. Figure 3 shows inhibition of DNA synthesis induced by IGF-I in Saos-2 cells by IGF-BP3. Part A shows the secretion of IGF-BP3 by induced EB1 cells. Part B shows that Saos-2 cells are sensitive to the activity of mitogenic IGF-I. Part C shows that p53-induced IGF-BP3, secreted from EB1 cells, inhibits the mitogenic activity of IGF-I. Figure 4a shows the expression of IGF-BP3 mRNA in human tissues. Figure 4b shows a working model that links p53 to the signaling pathway regulated by IGF. Figure 5a shows the induction of IGF-BP3 by DNA damage agents. Figure 5b shows that the induction of IGF-BP3 by DNA damage agents is dependent on p53. A new mechanism by which p53 regulates tumor growth is described herein. Specifically, it has been discovered that (1) the p53-sensitive DNA elements reside in the first and second introns of the IGF-BP3 gene; (2) wild type p53, but not mutant, induces an IGF-BP3 gene; and (3) this response is associated with an increase in both the synthesis and the secretion of IGF-BP3 in the extracellular space. The present discovery links p53 to the cytokine axis of IGF-I (and II) autocrine / paracrine / IGF receptor (IGFR) (see Figure 4b). IGF-BP3 binds IGFs and includes the interaction in their IGFRs, thereby acting as a growth inhibitor. Additionally, IGF-BP3 may have growth inhibitory effects unrelated to its ability to interact with IGF, but instead mediated by an IGFR-independent signaling pathway. In this way, IGF-BP3 can bind to two distinct signaling pathways associated with cell growth and inhibition.

The present discovery suggests that human tumors, in particular those with p53 mutations, can be treated by increasing or simulating IGF-BP3 functions. This method can employ IGF-BP3 modulators, which can be identified by methods described herein. These modulators up-regulate the expression or activity of IGF-BP3. One type of these modulators binds to one or both of the p53-sensitive elements of the IGF-BP3 (sequences A and B; SEC. ID. NOS .: 2 and 3, respectively). Another method employs the IGF-BP3 protein itself. In this method, the IGF-BP3 protein or recombinant protein is administered (for example, as supplied by UBI). For this method, the protein is produced, purified and formulated for administration by methods known in the art (for example, Tressel, T. J. et al. (1991), Biochem, Biophys., Res. Commun. 178: 625-33). Yet another method of treatment employs an expression vector comprising a nucleotide sequence encoding IGF-BP3. Suitable expression vectors include plasmids, but this invention includes other forms of expression vectors that now exist or become known in the art subsequently to the present. In addition, a useful expression vector typically contains the origin of replication, a promoter in the 5 'direction from the coding sequence, a transcription termination sequence. The expression vector may also include other DNA sequences known in the art, such as: stability guide sequences, which stabilize the expression product; secretory guide sequences, which allow the secretion of the expression product; environmental feedback sequences, which allow modulation of expression (for example, by the presence or absence of nutrients or other inducers in the growth medium; marker sequences, which allow phenotypic selection in transformed host cells; restriction sites; that allow cleavage by restriction endonucleases, and sequences that allow expression in several types of hosts, including prokaryotes, yeasts, fungi, plants and higher eukaryotes.The vector of cloning / expression directs the replication and expression of nucleic acids of the present invention, the suitable origins of replication include, for example, the Col El origins, the viral SV40 and the replication M13, the lacZ, the gal 10 promoter and the polyhedral promoter of the multiple nuclear polyhedrosis virus Autographa californica (AcMNPV) Suitable termination sequences include, for example, the hormone of cr bovine growth, SV40, lacZ and poly-acyl polyadenylation signals of AcMNPV. Examples of selectable markers include resistance to neomycin, ampicillin and hydromycin and the like. All of these materials are known in the art and are commercially available. Those skilled in the art can construct vectors having the above characteristics by recombinant DNA techniques, known in the art. See, Sambrook et al., Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989). Alternatively, they can use commercially available vectors that already incorporate these characteristics. Suitable commercially available vectors include the baculovirus expression vector pBlueBac, prokaryotic expression vectors pcDNAII and the yeast expression vector pYes2 (Invitrogen Corp., San Diego, CA).

In this method, the IGF-BP3 sequence can be under the control of a constitutive or inducible promoter. In the latter case, an inductor is co-administered. Suitable inducible promoters include mouse and dexamethasone breast tumor virus promoter, metallothionein and zinc promoter, yeast gal 4 promoter and galactose and the like. In addition, since IGF-I plays a role in apoptosis, inhibition of the IGF-I-IGF-IR axis could sensitize tumor cells to conventional cytotoxic agents by radiation and provide a new therapeutic approach to treatment of cancer. In this way, a cytotoxic agent or other anticancer agent can be co-administered as an additional step in the above methods. Suitable cytotoxic agents include paclitaxel, cisplatin, etoposide, paraplatin, bleomycin, plicamycin, doxorubicin, dimethyl-triazene-imidazole-carboxamide (DTIC), daunorubicin, cytarabine, procarbazine, 1- (β-chloroethyl) -1-nitrosourea (CCNU) , hydroxyurea, melphalan, 1,3-bis (ß-chloroet i 1) - 1-nitrosourea (BCNU), vincristine, vinblastine, o, p'-dichloro-diphenyldichloroethane (or, p'-DDD) (mitotane), cyclophosphamide , ifosfamide (a cyclophosphamide derivative), 5-fluorouracil, busulfan, dactinomycin, mitomycin-C, 6-thioguanine, thio-TEPA, chloroambucil, 6-mercaptopurine, methotrexate, nitrogen mustard, and the like. Other suitable anticancer agents include interferon, tamoxifen, testolactone, L-asparaginase, progesterone (egace, megestrol acetate), prednisone, androgens, estrogens, and the like. The above agents could be administered in approximately the dose and manner known in the art. Other suitable cytotoxic agents and other anticancer agents are listed in the "Orange Book" of the North American Food and Drug Administration, that is, in Approved Drug Products with Therapeutic Equivalence Evaluations, U.S. Dept. of Health and Human Services (1994), and its 1995 supplements. The invention further relates to methods for identifying substances useful in the treatment of tumors related to p53. One such method comprises introducing into a host cell (eg, by transfection), a construct having either or both of sequences A and Sequence B (SEQ ID NOS .: 2 and 3) operatively linked to an indicator gene As used in this context, the term "operably linked" means that the regulatory DNA sequences (SEQ ID NOS .: 2 and 3) are capable of increasing the expression of the RNA encoded by the reporter gene. The regulatory sequences may be in the 5 'direction of the coding region, in the 3' direction, or in an intron as in the gene for IGF-BP3. The reporter gene can be any number of indicators known in the art, such as luciferase, lacZ, chloraphenyl transferase (CAT), and the like. After introducing the construct into the host cell, the host cell can then be treated with the test substances. A test substance that binds to SEC. ID. NO .: 2 or 3 will up-regulate the expression of the indicator gene. This method identifies this substance as an IGF-BP3 modulator that can treat tumors related to p53. Another method for identifying substances useful in the treatment of tumors related to p53 takes advantage of the observation that p53-induced IGF-BP3 acts by inhibiting the synthesis of DNA induced by IGF (Figure 3). This method employs DNA (adenocin, thymidine, cytosine or guanidine) or nucleotide (ATP, GTP, TTP, or CTP) bases that have a detectable label. Suitable detectable labels include tritium-labeled thymidine (see, Figure 3 and Materials and Methods), 5'-bromo-2'-deoxyuridine and the like. In this method, a cell is treated with the labeled compound, a test substance, and IGF-I or IGF-II. For purposes of this method, "IGF-I" and "IGF-II" include recombinant variants thereof such as those provided by UBI. The synthesis of DNA dependent on IGF-I is inspected by the admission of the cell of the brand in the presence of variable concentrations of IGF and the test substance, as shown in Figure 3. If a test substance decreases IGF-dependent DNA synthesis, then it is useful in the treatment of tumors related to p53. Another method identifies compounds that simulate the activity of IGF-BP3 (for example, by inhibiting the binding of IGF to IGFR). In this method, cells or cell membrane preparations comprising IGFR with detectably labeled IGF (eg, radioiodinated IGF) are treated and detectable label binding is detected. This method can employ, for example, CHO cells that include IGFR or cell membrane preparations thereof. Ligand binding assays are well known in the art; see for example, Steele-Perkins et al. (1988), J. Biol. Chem. 263: 11486-92. The discoveries underlying the above methods have been made as follows: EB-1 colon carcinoma cells served as a model system for identifying new target genes induced by p53 that encode potential mediators of p53 tumor suppression. These cells carry a wild type, inducible p53 transgene under the control of the metallothionein promoter and undergo apoptosis in the induction of p53 by metal ions. Shaw et al. 81 ° 992), Proc. Nati Acad. Sci USA 89: 4495-9. A subtractive cDNA cloning approach was used (see Figure 1 and Materials and Methods), similar to the approach that was used to identify new p53 response genes in human Saos-2 osteosarcoma cells that contain a p53 coding transgene, Temperature sensitive, inducible and stably integrated. Buckbinder and collaborators (1994), Proc. Nati Acad. Sci. USA 91: 10640-4. This approach identified a number of fragments of CDNAs enriched and non-overlapping that were derived from different transcripts induced by p53, as determined by Northern blot analysis. The analysis of the nucleotide sequence determined that a particular fragment of cDNA was identical in sequence to a region in the gene 3 protein binding to the insulin-like growth factor, IGF-BP3. Cubbage et al. (1990), J ^ Biol. Chem. 265: 12642-9; access number to Genbank J05537, J05538. The Figure shows schematically the structure of the IGF-BP3 gene reported in Cubbage et al. (1990), as well as the location of the sequence of the isolated cDNA fragment (V9 probe). This fragment correlates to the 3 'untranslated region within exon 5. Figure Ib shows a Northern blot analysis of IBD-induced CdCl2 cells using the radiolabeled V9 probe to inspect the expression of IGF-BP3 mRNA. The induction of wt p53 is associated with a pronounced accumulation of mRNA levels of IGF-BP3, 10 hours after the addition of CdCl2. This induction (approximately 14 times) is comparable to that of other mRNAs encoded by previously characterized p53 response genes (p21, A28, and A26). See El-Deiry et al. (1993), Cell 75: 817-25; Buckbinder et al. (1994), Proc. Nati Acad. Sci. USA 91: 10640-4. Notably, the induction is specific to clonal EB-1 cells expressing p53; no induction was observed in the parental EB cells. The treatment of CdCl2 does not affect the levels of actin mRNA. The Figure shows a Northern blot analysis of the expression of mRNA and of 'IGF-BP3 in the clonal SAOS-2-d4h cells. These cells carry an inducible, temperature-sensitive transgene encoding human mutant p53V143A (described in detail in Buckbinder et al. (1994)). With the absence of tetracycline from the cell culture medium, the cells are expressed at high levels of the p53V143A protein. As shown, changing the cells to the allowable temperature of 30 ° C markedly induces the expression of IGF-BP3 mRNA. These findings confirm that the wild-type p53 that specifically induces the expression of a transcript of IGF-BP3 in a different cell type, while the p53 mutant does not induce IGF-BP3. Conscious of these findings, genomic resistance (eg, doxorubicin, ultraviolet light) that induces the expression of IGF-BP3 mRNA in normal diploid human fibroblasts (Figure 4a). Figure 1d shows a kinetic analysis of p53-mediated induction of IGF-BP3 mRNA expression in EB-1 cells. In comparison to the increase in mRNA levels of p21 and hdm-2, the induction of IGF-BP3 mRNA is somewhat delayed, increasing from 4 to 8 hours after stimulation with CdCl2. This raises the question whether the increase in expression of the IGF-BP3 gene represents a direct response of p53. In this way, a computer-based search for the DNA sequences in the IGF-BP3 gene related to the consensus binding site of p53 was carried out. EI-Deiry et al. 81992), Nature Genetics 1: 45-9. This search revealed two p53 binding sites, potentials in the first (Sequence A) and the second (Sequence B) introns of the IGF-BP3 gene, respectively (Figures la and 2a). As shown in Figure 2a, these binding sites are similar, but diverge by 2 or 3 nucleotides from the p53 consensus binding site. DNA binding analysis and EMSA analysis (Electro Mobility Shift Test) were also carried out using purified p53 human protein produced by baculovirus. Takenaka et al. (1995), J _; _ Biol. Chem. 270; 1-7. These analyzes confirm that Sequence A and Sequence B are p53 binding sites, specific (Figure 2). As shown in Figure 2b for the RGC binding site, the binding of wt p53 to the DNA sites of Sequence A and Sequence B is augmented by the addition of the C-terminal monoclonal antibody Pab421, which also produces the superchange. characteristic in the EMSA. Other p53 binding sites had similar results. Kern et al. (1991), Science 252: 1708-11. The binding is specific, as indicated by the ability of a wild-type, but non-mutant, p53 consensus DNA sequence to compete for the binding of p53 to either the DNA of Sequence A and the DNA of Sequence B The weaker binding of p53 to the DNA of Sequence B, in comparison to the DNA of Sequence A, is consistent with its weaker similarity to the consensus binding sequence of p53. Figure 2c shows that both Sequence A DNA and Sequence B confer inductibility to specific p53, wild-type to a heterologous promoter when introduced into human Saos-2 cells, confirming the nature of these DNA sequences as elements sensitive to p53. Consistent with DNA release binding studies, Sequence A confers a considerably stronger induction by p53 than Sequence B. However, two copies of Sequence B DNA confer increased sensitivity to p53, indicating that the DNA of the Sequence B, in comparison to the DNA of Sequence A, could potentially contribute to a p53-dependent induction of IGF-BP3 gene transcription. The induction of p53 of IGF-BP3 gene expression is significant because IGF-BP3 binds IGF-I and -II. Through this binding, IGF-BP3 reduces the availability of free IGF and thus regulates its proliferative and mitogenic effects. (For reviews, see Rechler (1993), Vitamins and Hormones 47: 1-114; Shimasaki, & Ling (1992), Prog. Growth Factor Res. 3: 243-66; Clemmons (1993), Mol. Reprod. Dev. 35: 368-75 and Baserga (1994), Cell 79: 927-30). Consistent with this regulation, p53-induced and recombinant IGF-BP3 was found to inhibit DNA synthesis induced by IGF-I in osteosarcoma Saos-2 cells (Figures 3a, 3b and 3c). These cells are the parental cells of the clonal D4H cells, in which it was found that P53 regulates the expression of IGF-BP3 (Figure 1). The addition of IGF-I (1 nM) to the Saos-3 cells stimulates DNA synthesis; as indicated by an increase in the incorporation of 3H-thymidine. The concomitant addition of purified recombinant IGF-BP3 (0-10 nM) inhibits DNA synthesis induced by IGF-I in a dose-dependent manner. The addition of IGF-BP3 alone does not inhibit the incorporation of 3H-thymidine, indicating that IGF-BP3 specifically inhibits the synthesis of DNA mediated by IGF-I in these cells. Both IGF-I and IGF-II act as autocrine and paracrine growth factors in adult tissues, affecting both normal and abnormal growth. Beserga (1994), Cell 79: 927-30; Goldring & Goldring (1991), Eukar. Gene Express .1: 301-21; Baserga et al. (1994), Adv. Exp. Med. Biol. 343: 105-12, Oh et al. (1993), Growth Reg. 3: 113-23. IGF-I / IGF-IR has been characterized particularly well. The loss of IGF-I and / or IGF-IR function is associated with: • cellular resistance to the mitogenic and transforming activities of the epidermal growth factor receptor; • resistance to the transformation activities of SV40 T antigen and SV40 T antigen and activated ras, combined; • apoptosis in vivo; • loss of growth of tumor cells in soft agar, syngeneic animals, and nude mice; and • immunogenic responses that can apparently lead to uniform regression of established homologous tumors. For reviews, see Beserga (1994), Cell 79: 927-30; and Baserga et al. (1994), Adv. Exp. Med. Biol. 343; 105-12, Recent reports suggest that 1 IGF-I protects the cell from p53-dependent apoptosis, induced by c-myc. Hermeking, & Eick (1994), Science 265: 2091-3; and Harrington and collaborators (1994); EMBO J. 13: 3286-95. In this way, IGF-I may act as a survival factor and may have a more pronounced factor in cells driven by oncogenes than in normal cells. Consequently, it is believed that IGF-BP3 plays an important autocrine and paracrine role in the control of growth by modulating the processes regulated by IGF. This role is especially significant because IGF-BP3 is expressed in multiple adult, human tissues (Figure 4a). The various experimental findings agree with this role for IGF-BP3: • IGF-BP3 inhibits DNA synthesis induced by IGF-I (Figure 3). • The cells that over express inhibit it in growth. Cohen et al. (1993), Mol. Endocrinol 7: 380-6. • The expression of IGF-BP3 is up-regulated in immobile and senescent cells (Moerman et al. (1993), Exp. Geronotol., 28: 361-70 and Grigoriev et al. (1994), J. Cell. Physiol. 160: 203 -eleven) . • The expression of IGF-BP3 is up-regulated in the arrest of the growth of estradiol-dependent breast cancer cells Pratt et al. (1993), Cancer Res. 53: 5193-8. Additionally, IGF-BP3 can regulate apoptosis by inhibiting IGF-I acting as a survival factor. In this report, it was shown that IGF-BP3 binds p53 to the IGF-I (II) / IGFR axis, providing insights into new, potential mechanisms, whereby p53 can regulate cell growth and apoptosis. Regulation of IGF-BP3 gene expression by wild-type p53 (Figure 1). Clonal parental EB and EB-1 cells were treated with or without CdCl2 (6 μM) for 10 hours. Poly (A) + RNA was isolated and Northern blots were prepared in quadruplicate. Transfers were entered with the cDNA V9 probe (IGF-BP3), with a cDNA probe for actin, and with cDNA probes for the transcripts regulated by p53, p21 / WAF1, A28 and A26, or actin, respectively, as previously described in Buckbinder et al. (1994), Proc. Nati Acad. Sci. USA 91: 10640-4. For Figure Ib, the subtraction procedure of the cDNA library based on PCR, described in Buckbinder et al. (1994) was used to identify the transcripts induced by wt p53 in the EB-1 cells activated by CdCl2 (6 μM, 8 hour stimulation). The promoter or stimulator DNA consisted of cDNA prepared from parental EB cells, treated with CdCl2, and untreated EB1 cells, as well as the cDNAs cloned for p53, p21, and hdm-2 to allow enrichment of the new cDNA sequences regulated. Buckbinder et al. (1994) determined the nucleotide sequence for clone V9 by automated DNA sequence analysis (ABI sequencer) and was found to be identical to one region (nucleotides 8095-8452) in the IGF-BP3 gene reported (Access No. of Genbank J05537). Buckbinder et al. (1994) describes the methods of RNA isolation and Northern blot analysis that was used. For Figure 1, Saos-2-D4H cells were cultured at 37 ° C without tetracycline to induce high levels of p53V143A protein expression. Subsequently these cells were incubated for 7 hours at 37 or 30 ° C (permissive temperature), as indicated. Northern blots were prepared with equal amounts of poly (A) + RNA and sequentially hybridized with radiolabeled V9 or GAPDH or 'cDNA probes. Characterization of DNA elements sensitive to and binding to p53 in the IGF-BP3 gene. A His-p53 fusion protein was produced in the baculovirus, purified, and the DNA binding reactions and EMSA analyzes were carried out following the procedures described in Takenaka et al. (1995), J ^ Biol. Chem. 270 : 1-7 Double-stranded DNA was used with the following sequences. Sequence A (SEQ ID NO: 2): 5 '-TCGAGAAAACAAGCCACCAACATGCTTGC-3' Sequence B (SEQ ID NO: 3): 5'-TCGAGAGGAGGGCAAGACCTGCCAAGCCTGGGTA-3 'consensus competitor (SEQ ID NO. : 5): 5 '-GATCTACCCAGGCTTGGCAGGTCTTGCCCTCCTC-3' mutant competitor (SEQ ID NO: 6): 0 5'-TCGAGCTTTGGACTTTTTCTGGCCA-3 'Luciferase indicator constructs were prepared by cloning the DNA of Sequence A and B in pUHC13 -3 as described in Buckbinder et al. (1994). The sequences were confirmed by automated DNA sequencing. The expression constructs of p53, pC53-SN3 and pC53-SCX3 (V143A), or the control pcDNA3 vector (0.5 μg) were co-transfected with a luciferase reporter plasmid (1.5 μg) in 3 × 10 5 Saos-2 cells using lipofectamine (Gibco BRL). Luciferase activity was determined as described in Buckbinder et al. (1994). For Figure 2a, EMSA was used to inspect the binding of the p53 protein produced by baculovirus, purified, described in Takenaka et al. (1995). Binding or binding reactions were performed in the presence of the monoclonal antibody Pab421 and the wild-type or mutant consensus p53 binding sites (200-fold molar excess), as indicated. For Figure 2c, constructs of the luciferase reporter were co-transfected with the expression constructs encoding wt p53 or p53V143A mutant, or the pcDNA vector, respectively. The luciferase constructs had either a copy of Sequence A, one or two copies of Sequence B, or multiple copies of the binding site of the bacterial tet repressor (pUHC13-3). Cells were collected after 16 hours and assessed for luciferase activity. Induction of DNA synthesis induced by IGF-I in Saos-2 - ^ IGF-BP3 cells (Figure 3). Sub-confluent cultures of human osteosarcoma Saos-2 cells were grown in enriched media (McCoy's medium supplemented with 15% fetal calf serum). These sub-confluent cultures were transfected to serum free Hams F12 medium (30 minutes) and then to F12 medium supplemented with 0.1% bovine serum albumin (BSA)., crystalline, Gibco BRL). These cultures were grown with or without recombinant IGF-I (1 nM, UBI) and with increasing amounts of recombinant IGF-BP3) or from the conditioned medium (CM) from treated EB or EB1 cells, as indicated. When the conditioned medium was used, it was dialyzed against the Hams F12 medium to remove the CdCl2 and the filter was sterilized. In some cases, the conditioned medium of IGF-BP3 was depleted by immuno-depletion using an IGF-BP3 monoclonal antibody (Accurate Scientific) or the control MDM2 antibody (Oncogene Science). After 18 hours of incubation, the cells were boosted with 3 H-thymidine (2 μCi / mL) for 3 hours. The cells were washed in phosphate buffered saline (PBS, pH 7.4). The liquid scintillation count was used to measure the incorporation of 3H-thymidine into acid-soluble material. The average accounts ± S.D. from triplicate cultures are shown. Multi-tissue expression of IGF-BP3 mRNA (Figure 4). Northern blot with poly (A) + RNA (2 μg / lane) was used from adult, human, multiple tissues (Clontech). These blots were hybridized with the radiolabeled V9 probe (IGF-BP3).

It is noted that in relation to this date, the best method known to the applicant to carry out the present invention is that which is clear from the present description of the invention.

Having described the invention as above, the contents of the following are claimed as property:

Claims (11)

1. A method for treating tumors related to p53, characterized in that it comprises administering an effective amount of an IGF-BP3 modulator, wherein the modulator up-regulates the expression or activity of IGF-BP3.

2. The method according to claim 1, characterized in that the modulator is linked to the SEQ. ID. DO NOT. 2 or 3, in genomic DNA encoding IGF-BP3.

3. A method for treating tumors related to p53, characterized in that it comprises administering an effective amount of IGF-BP34.

A method for treating tumors related to p53, characterized in that it comprises administering an effective amount of an expression vector comprising a nucleotide sequence encoding IGF-BP3.

5. The method according to claim 4, characterized in that (a) the nucleotide sequence encoding IGF-BP3 is operably linked to an inducible promoter or an inducible enhancer; and (b) the method further comprises administering an inducer capable of up-regulating or initiating expression of the protein.

6. The method according to any of claims 1, 3, 4 or 5, characterized in that the method further comprises administering a cytotoxic agent.

7. The method according to claim 6, characterized in that the cytotoxic agent is selected from the group consisting of paclitaxel, cisplatin, doxorubicin etoposide, camptothecin, mitomycin-C, cyclophosphamide, and methotrexate.

8. A method for treating tumors related to p53, characterized in that it comprises inhibiting the binding of IGF-I or IGF-II to IGFR.

9. A method for identifying a substance useful in the treatment of tumors related to p53, characterized in that it comprises: (a) applying a test substance to a cell having an expression vector comprising (i) a nucleotide sequence for a reporter gene , which is operably linked to (ii) the nucleotide sequence of SEC. ID. NOS .: 2 or 3 or both; and (b) analyzing the cell to detect the expression of the reporter gene; wherein the expression of the reporter gene indicates that the test substance upregulates IGF-BP3 and is also useful in the treatment of tumors related to p53.

10. A method for detecting a substance useful in the treatment of tumors related to p53, characterized in that it comprises: (a) treating a tumor cell comprising the IGF-I or IGF-II receptor with (i) a test substance, ( ii) a DNA base or nucleotide having a detectable label, and (iii) IGF-I or IGF-II; and (b) detecting the admission of the detectable label in the cells or the presence of invariant concentration of IGF-I or IGF-II and the test substance; wherein a decrease in the admission of the detectable label in the presence of the test substance indicates that the test substance inhibits the synthesis of DNA directed by IGF and thus is useful in the treatment of tumors related to p53.

11. A method for detecting a substance useful in the treatment of tumors related to p53, characterized in that it comprises: (a) treating a cell or cell membrane preparation comprising an IGF receptor: (i) a test substance, and (ii) ) IGF-I or IGF-II having the detectable label; and (b) detecting the binding of the detectable label in the preparation of the cells or the cell membrane in the presence of the test substance; wherein a decrease in the admission of the detectable label in the presence of the test substance indicates that the test substance inhibits the binding of IGF-I or IGF-II to the receptor.